Lyophilized formulations for factor xa antidote

ABSTRACT

The present disclosure relates to solutions and methods of preparing lyophilized formulations of factor Xa (fXa) antidotes. A suitable aqueous formulation suitable for lyophilization can include a fXa antidote, a solubilizing agent, a stabilizer, and a crystalline component, wherein the formulation does not collapse during lyophilization.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of International Application No.PCT/US2015/046173, filed Aug. 20, 2015, which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Application 62/039,809, filedAug. 20, 2014, the content of each of which is incorporated herein byreference in its entireties and for all purposes.

BACKGROUND

Anticoagulants serve a need in the marketplace in treatment orprevention of undesired thrombosis in patients with a tendency to formblood clots, such as, for example, those patients having clottingdisorders, confined to periods of immobility or undergoing medicalsurgeries. One of the major limitations of anticoagulant therapy,however, is the bleeding risk associated with the treatments, andlimitations on the ability to rapidly reverse the anticoagulant activityin case of overdosing or if an urgent surgical procedure is required.Thus, specific and effective antidotes to all forms of anticoagulanttherapy are highly desirable.

Delivery of biologically active proteins by injection is generally thedelivery route of choice when oral delivery is not practical or animmediate therapeutic activity is required. However, biological,chemical, and physical barriers such as poor long-term storage,osmolality, solubility, and stability make delivery of biologicallyactive agents by injection to mammals problematic. Lyophilization cansolve long-term storage issues. Nevertheless, there are problems thatalso occur with lyophilization, such as poor solubility and stability ofthe lyophilate. Therefore, there exists a need for improved injectablepreparations of antidotes to anticoagulants, which are stable andsoluble. The disclosure satisfies these and other needs.

Any and all publications, patents, patent applications mentioned hereinare hereby incorporated by reference in their entirety.

SUMMARY

The present disclosure provides lyophilized formulations for aderivative of the factor Xa (fXa) protein, referred to as the“r-Antidote.” Compared to the wild-type fXa protein, the r-Antidote hasmodifications to the Gla domain and the active site, retains fXa'sability to bind to a fXa inhibitor but does not assemble into aprothrombinase complex. The r-Antidote is a two-chain polypeptide (seeSEQ ID NO. 3 in Table 3, which includes a light chain (SEQ ID NO. 4) anda heavy chain (SEQ ID NO. 5) connected with a single disulfide bondbetween Cysteine 98 (Cys98) of the light chain and Cysteine 108 (Cys108)of the heavy chain.

Also like the wild-type fXa, the r-Antidote undergoes post-translationalmodifications resulting in glycosylation at certain amino acid residues,e.g., Ser56, Ser72, Ser76 and Thr82 of the light chain and Thr249 of theheavy chain, and a modified residue, (3R)-3-hydroxyAsp at Asp29 of thelight chain. Further, in addition to the inter-chain disulfide bond,there are intra-chain disulfide bonds formed between Cysteines 16 and27, 21 and 36, 38 and 47, 55 and 66, 62 and 75, and 77 and 90 of thelight chain, and between Cysteines 7 and 12, 27 and 43, 156 and 170, and181 and 209 of the heavy chain.

Given the two-chain structure and various post-translationalmodifications of the r-Antidote, it is shown herein that development ofa stable lyophilized formulation that provides a stable and solublesolution with an acceptable osmolality presents a great challenge.Unexpectedly, however, the present inventors were able to arrive at asolution that balances protein solubility, stability, cake structure andosmolality.

In one embodiment, the present disclosure provides an aqueousformulation. In one embodiment, the formulation comprises from 10 mM to55 mM arginine or from 8 mM to 35 mM citrate, from 1% to 3% sucrose(w/v), from 2% to 8% mannitol (w/v) and at least 5 mg/mL of a two-chainpolypeptide comprising a first chain of the amino acid sequence of SEQID NO. 4, a second chain of the amino acid sequence of SEQ ID NO. 5, anda disulfide bond between a first Cysteine residue at position 98 (Cys98)of SEQ ID NO. 4 and a second Cysteine residue at position 108 (Cys108)of SEQ ID NO. 5, wherein the formulation has a pH from 7.5 to 8.

In some aspects, the formulation comprises from 40 mM to 50 mM arginine,from 1.5% to 2.5% sucrose (w/v), from 4.5% to 5.5% mannitol (w/v) and atleast 10 mg/mL of the polypeptide. In some aspects, the formulationcomprises from 10 mM to 30 mM citrate, from 1.5% to 2.5% sucrose (w/v),from 4.5% to 5.5% mannitol (w/v) and at least 10 mg/mL of thepolypeptide.

In some aspects, the formulation comprises from 40 mM to 50 mM arginine,from 1.5% to 2.5% sucrose (w/v), from 4.5% to 5.5% mannitol (w/v) and atleast 18, 19 or 20 mg/mL of the polypeptide. In some aspects, theformulation comprises from 10 mM to 30 mM citrate, from 1.5% to 2.5%sucrose (w/v), from 4.5% to 5.5% mannitol (w/v) and at least 10 mg/mL ofthe polypeptide.

In some aspects, the formulation comprises about 45 mM arginine, about2% sucrose (w/v), about 5% mannitol (w/v) and about 10 mg/mL of atwo-chain polypeptide comprising a first chain comprising the amino acidsequence of SEQ ID NO. 4, a second chain comprising the amino acidsequence of SEQ ID NO. 5, and a disulfide bond between a first Cysteineresidue at position 98 (Cys98) of SEQ ID NO. 4 and a second Cysteineresidue at position 108 (Cys108) of SEQ ID NO. 5, wherein theformulation has a pH of about 7.8. In one aspect, the formulationfurther includes polysorbate 80 (0.01% w/v to 0.02% w/v) and/or abuffer.

In some aspects, the formulation comprises about 45 mM arginine, about2% sucrose (w/v), about 5% mannitol (w/v) and about 20 mg/mL of atwo-chain polypeptide comprising a first chain comprising the amino acidsequence of SEQ ID NO. 4, a second chain comprising the amino acidsequence of SEQ ID NO. 5, and a disulfide bond between a first Cysteineresidue at position 98 (Cys98) of SEQ ID NO. 4 and a second Cysteineresidue at position 108 (Cys108) of SEQ ID NO. 5, wherein theformulation has a pH of about 7.8. In one aspect, the formulationfurther includes polysorbate 80 (0.01% w/v to 0.02% w/v) and/or abuffer.

In some aspects, the polypeptide comprises an amino acid residue that ismodified to be different from natural amino acids. In some aspects,residue Asp29 of the first chain is modified to (3R)-3-hydroxyAsp atAsp29. In some aspects, the polypeptide comprises at least anintra-chain disulfide bond for each of the first and second chains.

Also provided, in one embodiment, is a method of preparing a lyophilizedformulation of a two-chain polypeptide comprising a first chain of theamino acid sequence of SEQ ID NO. 4, a second chain of the amino acidsequence of SEQ ID NO. 5, and a disulfide bond between a first Cysteineresidue at position 98 (Cys98) of SEQ ID NO. 4 and a second Cysteineresidue at position 108 (Cys108) of SEQ ID NO. 5, comprisinglyophilizing the aqueous formulation as describe above.

Another embodiment provides a lyophilized composition prepared bylyophilizing the aqueous formulation of the present disclosure.

In one embodiment, the present disclosure provides a lyophilizedcomposition comprising at least 10% (w/w) of a two-chain polypeptidecomprising a first chain of the amino acid sequence of SEQ ID NO. 4, asecond chain of the amino acid sequence of SEQ ID NO. 5, and a disulfidebond between a first Cysteine residue at position 98 (Cys98) of SEQ IDNO. 4 and a second Cysteine residue at position 108 (Cys108) of SEQ IDNO. 5, and arginine:sucrose:mannitol in a weight ratio of the range(0.6-0.95):(1-3):(2-8), or alternatively L-arginine HCl:sucrose:mannitolin a weight ratio of the range (0.5-1.4):(1-3):(2-8).

In some aspects, the lyophilized composition comprises at least 15%,16%, 17%, 18% or 19% (w/w) of the two-chain polypeptide. In someaspects, the weight ratio of L-arginine HCl:sucrose:mannitol is in therange of (0.9-1):(1.5-2.5):(4.5-5.5).

Also provided is a lyophilized composition comprising at least 10% (w/w)of a two-chain polypeptide comprising a first chain of the amino acidsequence of SEQ ID NO. 4, a second chain of the amino acid sequence ofSEQ ID NO. 5, and a disulfide bond between a first Cysteine residue atposition 98 (Cys98) of SEQ ID NO. 4 and a second Cysteine residue atposition 108 (Cys108) of SEQ ID NO. 5, and citrate:sucrose:mannitol in aweight ratio of the range (0.15-0.66):(1-3):(2-8). In some aspects, thelyophilized composition comprises at least 10%, 15%, 16%, 17%, 18%, or19% (w/w) of the two-chain polypeptide. In some aspects, the weightratio of citrate:sucrose:mannitol is in the range of(0.19-0.57):(1.5-2.5):(4.5-5.5).

Another embodiment of the present disclosure provides a solutionprepared by dissolving the lyophilized composition of the disclosure. Insome aspects, the solvent is water or saline.

Yet another embodiment provides a method of reducing bleeding in asubject undergoing anticoagulant therapy with a factor Xa inhibitorcomprising administering to the subject an effective amount of asolution of the disclosure. In some aspects, the factor Xa inhibitor isapixaban, rivaroxaban or betrixaban.

Still, also provided, in one embodiment, is an aqueous formulation,comprising a polypeptide comprising the amino acid sequence of SEQ IDNO. 3 or an amino acid sequence having at least 95% sequence identity toSEQ ID NO. 3, a solubilizing agent, a stabilizer, and a crystallinecomponent, wherein the formulation does not collapse duringlyophilization.

In some aspects, the crystalline component is mannitol. In some aspects,the mannitol is present in a concentration from 2% to 8% (w/v). In someaspects, the solubilizing agent is arginine or citrate and thestabilizer is sucrose. In some aspects, the aqueous formulation furthercomprises a surfactant and a buffer. In some embodiments, provided is alyophilized composition prepared by lyophilizing the aqueousformulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F are charts showing the solubility of the r-Antidote underdifferent conditions (pH, solubilizing agent, ionic strength). Shadedbars indicate that protein precipitation was observed and empty barsindicate that no protein precipitation was observed.

FIG. 2 is a DSC heat flow thermogram for 10 mm tris solution showingcooling at 1° C./min and the crystallization exotherm for tris at −32°C.

FIG. 3 is a DSC thermogram during cooling of 10 mm tris with 95 mmarginine. No crystallization exotherm for tris.

FIG. 4 is a DSC thermogram for a solution containing 10 mm tris, 2%sucrose, and 2% mannitol showing crystallization of mannitol atapproximately −18° C.

FIG. 5 is a DSC thermogram for a solution of 10 mm tris, 95 mm arginine,2% sucrose, and 2% mannitol showing the tg′ for sucrose at −42° C.solution was annealed for 5 hours at −20° C.

FIG. 6 is a DSC thermogram for a solution of 10 mm tris, 95 mm arginine,2% sucrose, and 2% mannitol showing the annealing step at −20° C. for 5hours with no evidence of a crystallization exotherm.

FIG. 7 is a DSC thermogram for 10 mm sodium phosphate solution showing acrystallization exotherm for sodium phosphate at approximately −10° C.

FIG. 8 is a DSC non-reversing heat flow thermogram for 10 mm sodiumphosphate with 2% sucrose and 2% mannitol showing a crystallizationexotherm with an onset at approximately −33° C.

FIG. 9 is a DSC heat flow thermogram for 10 mm sodium phosphate, 95 mmarginine, 2% sucrose, and 2% mannitol exhibiting no thermal eventsbesides the ice melting endotherm.

FIG. 10 is a DSC heat flow thermogram for 10 mm tris, 10 mm citrate, 2%sucrose, and 5% mannitol showing a crystallization exotherm with anonset of approximately 24 minutes at −25° C.

FIG. 11 is a DSC heat flow thermogram for 10 mm tris, 20 mm citrate, 2%sucrose, and 5% mannitol showing a crystallization exotherm with anonset of approximately 30 minutes at −25° C.

FIG. 12 is a UV concentration data for tris and phosphate solutionformulations stored at 5° C. for up to 2 weeks compared with lyophilizedformulations at T0.

FIG. 13 is a UV concentration data for tris and phosphate solutionformulations stored at 25° C. for up to 2 weeks compared withlyophilized formulations at T0.

FIG. 14 is a UV concentration data for tris and phosphate lyophilizedformulations stored at 25° C. compared with solution formulations at T0.

FIG. 15 is a DSC thermogram for 10 mm tris, 9.5 mm arginine, 2% sucrose,2% mannitol, and 0.01% PS80 formulation showing the onset ofcrystallization of mannitol at 70 minutes (onset time of annealing) at−22° C.

FIG. 16 is a DSC thermogram for 10 mm tris, 47.5 mm arginine, 2%sucrose, 4% mannitol, and 0.01% PS80 formulation showing a the onset ofcrystallization of mannitol at 30 minutes at −25° C.

FIG. 17 is a DSC crystallization exotherm for mannitol when the 10 mmtris, 47.5 mm arginine, 2% sucrose, 5% mannitol, and 0.01% PS80 solutionis cooled at 1° C./min to −40° C.

FIG. 18 shows DSC crystallization exotherm for mannitol when the 10 mmtris, 47.5 mm arginine, 2% sucrose, 5% mannitol, and 0.01% PS80 solutionis annealed at −25° C. the onset of crystallization is approximately 23minutes.

DETAILED DESCRIPTION I. Definitions

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 10%. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about”. It also is to be understood, althoughnot always explicitly stated, that the reagents described herein aremerely exemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a pharmaceutically acceptable carrier”includes a plurality of pharmaceutically acceptable carriers, includingmixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this disclosure.Embodiments defined by each of these transition terms are within thescope of this disclosure.

The term “protein” and “polypeptide” are used interchangeably and intheir broadest sense to refer to a compound of two or more subunit aminoacids, amino acid analogs or peptidomimetics. The subunits may be linkedby peptide bonds. In another embodiment, the subunit may be linked byother bonds, e.g., ester, ether, etc. A protein or peptide must containat least two amino acids and no limitation is placed on the maximumnumber of amino acids which may comprise a protein's or peptide'ssequence. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D and L optical isomers, amino acid analogs and peptidomimetics.Single letter and three letter abbreviations of the naturally occurringamino acids are listed below.

“Factor Xa” or “fXa” or “fXa protein” is a serine protease in the bloodcoagulation pathway, which is produced from the inactive factor X (fX,SEQ ID NO. 1, Table 1). The nucleotide sequence coding human factor X(“fX”) can be found in GenBank with accession number “NM_000504.” Uponcatalytic cleavage of the first 52 residues of the heavy chain, fX isactivated to fXa. FXa contains a light chain and a heavy chain. Thefirst 45 amino acid residues (residues 1-45 of SEQ ID NO. 1) of thelight chain is called the Gla domain because it contains 11post-translationally modified γ-carboxyglutamic acid residues (Gla). Italso contains a short (6 amino acid residues) aromatic stack sequence(residues 40-45 of SEQ ID NO. 1). Chymotrypsin digestion selectivelyremoves the 1-44 residues resulting in Gla-domainless fXa. The serineprotease catalytic domain of fXa locates at the C-terminal heavy chain.The heavy chain of fXa is highly homologous to other serine proteasessuch as thrombin, trypsin, and activated protein C.

“Native fXa” or “wild-type fXa” refers to the fXa naturally present inplasma or being isolated in its original, unmodified form, whichprocesses the biological activity of activating prothrombin thereforepromoting formation of blood clot. The term includes naturally occurringpolypeptides isolated from tissue samples as well as recombinantlyproduced fXa. “Active fXa” refers to fXa having the procoagulantactivity of activating prothrombin. “Active fXa” may be a native fXa ormodified fXa that retains procoagulant activity.

As used herein, “fXa derivatives” refer to modified fXa proteins that donot compete with fXa in assembling into the prothrombinase complex andhave reduced or no procoagulant or catalytic activities, and yet bindand/or substantially neutralize the anticoagulants, such as fXainhibitors. “Procoagulant activity” of an fXa protein or fXa derivative,in some aspects, refers to the enzymatic activity that the wild-typeactive fXa polypeptide carries. Examples of fXa derivatives are providedin U.S. Pat. No. 8,153,590, and PCT publications W)2009/042962 andWO2010/056765, and further provided herein, such as SEQ ID NO: 2 and 3and biological equivalents thereof.

The “enzymatic activity” of an fXa polypeptide or derivatives thereofrefers to the polypeptide's ability to catalyze a biochemical reactionwith a substrate through direct interaction with the substrate.

SEQ ID NO: 2 contains 3 mutations relative to the wild type fXa. Thefirst mutation is the deletion of 6-39 aa in the Gla-domain of fX. Thesecond mutation is replacing the activation peptide sequence 143-194 aawith -RKR-. This produces a -RKRRKR- (SEQ ID NO: 6) linker connectingthe light chain (SEQ ID NO: 4) and the heavy chain (SEQ ID NO: 5). Uponsecretion, this linker is cleaved resulting in a two-chain polypeptide,SEQ ID NO: 3 (r-Antidote). The third mutation is mutation of active siteresidue S379 to an Ala residue. This amino acid substitution correspondsto amino acid 296 and 290 of SEQ ID NOS: 1 and 3, respectively.

The term “r-Antidote” refers to a processed two-chain polypeptideprocessing product of SEQ ID NO: 2, after cleavage of the linker. Thisis represented by SEQ ID NO: 3. The r-antidote is disclosed in, e.g.,U.S. Pat. No. 8,153,590, the content of which is incorporated to thepresent disclosure by reference. The r-Antidote includes a light chain(SEQ ID NO. 4) and a heavy chain (SEQ ID NO. 5) connected with a singledisulfide bond between Cysteine 98 (Cys98) of the light chain andCysteine 108 (Cys108) of the heavy chain. Like the wild-type fXa, incertain production batches, the r-Antidote undergoes post-translationalmodifications resulting in glycosylation at certain amino acid residues,e.g., Ser56, Ser72, Ser76 and Thr82 of the light chain and Thr249 of theheavy chain, and a modified residue, (3R)-3-hydroxyAsp at Asp29 of thelight chain. Further, in addition to the inter-chain disulfide bond,there can be intra-chain disulfide bonds formed between Cysteines 16 and27, 21 and 36, 38 and 47, 55 and 66, 62 and 75, and 77 and 90 of thelight chain, and between Cysteines 7 and 12, 27 and 43, 156 and 170, and181 and 209 of the heavy chain.

TABLE 1 Polypeptide Sequence of Inactive Human Factor X (SEQ ID NO: 1)  1 ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN121 GKACIPTGPY PCGKQTLERR KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF181 NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ241 AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP301 ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ361 NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK421 WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 2 Polypeptide Sequence of the r-Antidote prior to removal of the-RKRRKR- (SEQ ID NO. 6) linker (SEQ ID NO: 2) Light Chain (SEQ ID NO: 4)  1 ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN121 GKACIPTGPY PCGKQTLER Linker (SEQ ID NO: 6) RKRRKRHeavy Chain (SEQ ID NO: 5) 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 3 Polypeptide Sequence ofa Human Factor Xa triple mutant afterremoval of the -RKRRKR-(SEQ ID NO. 6) linker (SEQ ID NO: 3)Light Chain (SEQ ID NO: 4)   1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK  61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain (SEQ ID NO: 5) 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

The present disclosure also provides a variety of biological equivalentsof r-Antidote (or their precursors, represented by SEQ ID NO: 2), oralternatively polypeptides having certain sequence identity to SEQ IDNO: 3. In one aspect, such biological equivalents retain the structuralcharacteristics of SEQ ID NO: 3, that is, a modified active site and adeleted or modified Gla domain. In another aspect, such biologicalequivalents retain the functional features of SEQ ID NO: 3, that is, notcompeting with fXa in assembling into the prothrombinase complex andhaving reduced or no procoagulant (e.g., enzymatic or catalytic)activities.

The term “active site” refers to the part of an enzyme or antibody wherea chemical reaction occurs. A “modified active site” is an active sitethat has been modified structurally to provide the active site withincreased or decreased chemical reactivity or specificity. Examples ofactive sites include, but are not limited to, the catalytic domain ofhuman factor X comprising the 235-488 amino acid residues, and thecatalytic domain of human factor Xa comprising the 195-448 amino acidresidues. Examples of modified active site include, but are not limitedto, the catalytic domain of human factor Xa comprising 195-448 aminoacid residues in SEQ ID NO: 1 with at least one amino acid substitutionat position Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term “lyophilized formulation” refers to a pharmaceuticalformulation or composition comprising a polypeptide of interest that isfreeze-dried.

As used herein, the term “bulking agent” refers to an ingredient thatprovides bulk to the lyophilized formulation. Examples of bulking agentsinclude, without limitation, mannitol, trehalose, lactose, sucrose,polyvinyl pyrrolidone, sucrose, glucose, glycine, cyclodextrins,dextran, solid PEGs and derivatives and mixtures thereof. In oneembodiment, a formulation of the present disclosure optionally includesa bulking agent.

As used herein, a “pharmaceutically acceptable cake” refers to anon-collapsed solid drug product remaining after lyophilization that hascertain desirable characteristics, e.g. pharmaceutically acceptable,long-term stability, a short reconstitution time, an elegant appearanceand maintenance of the characteristics of the original solution uponreconstitution. The pharmaceutically acceptable cake can be solid,powder or granular material. The pharmaceutically acceptable cake mayalso contain up to five percent water by weight of the cake.

As used herein, the term “lyophilization” or freeze drying refers to aprocess in which water is removed from a product after it is frozen andplaced under a vacuum, allowing the ice to change directly from solid tovapor without passing through a liquid phase. The process consists ofthree separate, unique, and interdependent processes; freezing, primarydrying (sublimation), and secondary drying (desorption). Methods forlyophilizing polypeptides used in this disclosure are described hereinand well known in the art.

The term “buffer” as used herein denotes a pharmaceutically acceptableexcipient, which stabilizes the pH of a pharmaceutical preparation.Suitable buffers are well known in the art and can be found in theliterature. Pharmaceutically acceptable buffers comprise but are notlimited to tris-buffers, arginine-buffers, histidine-buffers,citrate-buffers, succinate-buffers and phosphate-buffers. Independentlyfrom the buffer used, the pH can be adjusted with an acid or a baseknown in the art, e.g., succinic acid, hydrochloric acid, acetic acid,phosphoric acid, sulfuric acid and citric acid, succinate, citrate, trisbase, histidine, histidine HCl, sodium hydroxide and potassiumhydroxide. Suitable buffers include, without limitation, histidinebuffer, 2-morpholinoethanesulfonic acid (MES), cacodylate, phosphate,acetate, succinate, and citrate. The concentration of the buffer can bebetween about 4 mM and about 60 mM, or alternatively about 4 mM to about40 mM, or alternatively about 5 mM to about 25 mM.

“Cryoprotectants” are known in the art and include without limitation,e.g., sucrose, trehalose, and glycerol. A cryoprotectant exhibiting lowtoxicity in biological systems is generally used.

The term “tonicity agent” as used herein denotes pharmaceuticallyacceptable agents used to modulate the tonicity of the formulation.Isotonicity generally relates to the osmotic pressure relative to asolution, usually relative to that of human blood serum. A formulationcan be hypotonic, isotonic or hypertonic. In one aspect, the formulationis isotonic. An isotonic formulation is liquid or liquid reconstitutedfrom a solid form, e.g. from a lyophilized form and denotes a solutionhaving the same tonicity as some other solution with which it iscompared, such as physiologic salt solution and the blood serum.Suitable isotonicity agents include but are not limited to sodiumchloride, potassium chloride, glycerin and any component from the groupof amino acids, sugars, as defined herein as well as combinationsthereof.

As used herein, the term “surfactant” refers to a pharmaceuticallyacceptable organic substance having amphipathic structures; namely, itis composed of groups of opposing solubility tendencies, typically anoil-soluble hydrocarbon chain and a water-soluble ionic group.Surfactants can be classified, depending on the charge of thesurface-active moiety, into anionic, cationic, and nonionic surfactants.Surfactants are often used as wetting, emulsifying, solubilizing, anddispersing agents for various pharmaceutical compositions andpreparations of biological materials. In some embodiments of thepharmaceutical formulations described herein, the amount of surfactantis described as a percentage expressed in weight/volume percent (w/v %).Suitable pharmaceutically acceptable surfactants include but are notlimited to the group of polyoxyethylensorbitan fatty acid esters(Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethyleneethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer(Poloxamer, Pluronic), or sodium dodecyl sulphate (SDS).Polyoxyethylenesorbitan-fatty acid esters include polysorbate 20, (soldunder the trademark Tween 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Polyethylene-polypropylene copolymers includethose sold under the names Pluronic® F68 or Poloxamer 188™.Polyoxyethylene alkyl ethers include those sold under the trademarkBrij™. Alkylphenolpolyoxyethylene ethers include those sold under thetradename Triton-X.

A “lyoprotectant” refers to a pharmaceutically acceptable substance thatstabilizes a protein during lyophilization (the process of rapidfreezing and drying in a high vacuum). Examples of lyoprotectantsinclude, without limitation, sucrose, trehalose or mannitol.

An “antioxidant” refers to a molecule capable of slowing or preventingthe oxidation of other molecules. Oxidation is a chemical reaction thattransfers electrons from a substance to an oxidizing agent. Oxidationreactions can produce free radicals, which start chain reactions thatdestabilize the protein therapeutics and ultimately affect the productactivity. Antioxidants terminate these chain reactions by removing freeradical intermediates, and inhibit other oxidation reactions by beingoxidized themselves. As a result, antioxidants are often reducingagents, chelating agent and oxygen scavengers such as citrate, EDTA,DPTA, thiols, ascorbic acid or polyphenols. Non-limiting examples ofantioxidants include ascorbic acid (AA, E300), thiosulfate, methionine,tocopherols (E306), propyl gallate (PG, E310), tertiarybutylhydroquinone (TBHQ), butylated hydroxyanisole (BHA, E320) andbutylated hydroxytoluene (BHT, E321).

A “preservative” is a natural or synthetic chemical that is added toproducts such as foods, pharmaceuticals, paints, biological samples,wood, etc. to prevent decomposition by microbial growth or byundesirable chemical changes. Preservative additives can be used aloneor in conjunction with other methods of preservation. Preservatives maybe antimicrobial preservatives, which inhibit the growth of bacteria andfungi, or antioxidants such as oxygen absorbers, which inhibit theoxidation of constituents. Common antimicrobial preservatives include,benzalkonium chloride, benzoic acid, cholorohexidine, glycerin, phenol,potassium sorbate, thimerosal, sulfites (sulfur dioxide, sodiumbisulfite, potassium hydrogen sulfite, etc.) and disodium EDTA. Otherpreservatives include those commonly used in patenteral proteins such asbenzyl alcohol, phenol, m-cresol, chlorobutanol or methylparaben.

The term “surfactant” as used herein means compounds that lower thesurface tension (or interfacial tension) between two liquids or betweena liquid and a solid. Surfactants may act as detergents, wetting agents,emulsifiers, foaming agents, and dispersants.

Examples of surfactants include polysorbate 80, fatty acid and alkylsulfonates; benzethanium chloride, e.g., HY AMINE 1622 from Lonza, Inc.(Fairlawn, N.J.); polyoxyethylene sorbitan fatty acid esters, e.g., theTWEEN Series from Uniqema (Wilmington, Del.); and natural surfactants,such as sodium taurocholic acid,1-palmitoyl-2-Sn-glycero-3-phosphocholine, lecithin and otherphospholipids. Such surfactants, e.g., minimize aggregation oflyophilized particles during reconstitution of the product. Thesesurfactants may comprise from about 0.001% to about 5% w/v.

II. Formulations

As provided, the wild-type fXa is a two-chain polypeptide. So are manyforms of fXa antidotes, including the r-Antidote (SEQ ID NO: 3), whichincludes a light chain (SEQ ID NO. 4) and a heavy chain (SEQ ID NO. 5)connected with a single disulfide bond between Cysteine 98 (Cys98) ofthe light chain and Cysteine 108 (Cys108) of the heavy chain. Also likethe wild-type fXa, the r-Antidote expressed in cells undergoespost-translational modifications resulting in glycosylation at certainamino acid residues, e.g., Ser56, Ser72, Ser76 and Thr82 of the lightchain and Thr249 of the heavy chain, and a modified residue,(3R)-3-hydroxyAsp at Asp29 of the light chain. Further, in addition tothe inter-chain disulfide bond, there can be one or more intra-chaindisulfide bonds formed between Cysteines 16 and 27, 21 and 36, 38 and47, 55 and 66, 62 and 75, and 77 and 90 of the light chain, and betweenCysteines 7 and 12, 27 and 43, 156 and 170, and 181 and 209 of the heavychain.

Given the two-chain structure and various post-translationalmodifications of the fXa antidotes, it is shown herein that developmentof a stable lyophilized formulation that provides a stable and solublesolution with an acceptable osmolality presents a great challenge.

Using the r-Antidote as an example, experimental data showed that a highconcentration of a solubilizing agent is required to maintain areasonable solubility for the r-Antidote. In particular, the solubilitystudies in Example 4 shows that both citrate and arginine significantlyincrease the solubility of the r-Antidote. Further, the examples showedthat the r-Antidote could remain soluble in the solution when theconcentration of arginine was at 95 mM, or at least 10 mM.

Further, during the lyophilization process, it was determined that thetemperature of the protein needed to be maintained below the determinedcollapse temperature (about −40° C.) to obtain acceptable lyophilizedsamples (Example 6). Maintaining such low product temperature, however,is not feasible in practice. Therefore, the data demonstrate that acrystallizing component (e.g., mannitol) is required to serve as ascaffold that can hold the amorphous protein material in place duringand after freeze drying.

It was further discovered, however, the presence of a high concentrationof arginine (e.g., 95 mM) prevented crystallization of mannitol (Example7). Meanwhile, the presence of mannitol increases the totalconcentration of sugar in the formulation, leading to unacceptableosmolality of the solution (Example 7).

Development of a suitable lyophilized formation for the r-Antidote,therefore, had conflicting requirements for the concentration ofarginine as a solubilizing agent, mannitol as a crystallizing agent, andsucrose as a stabilizing agent. It was, at the best, unpredictablewhether such requirements could be balanced to generate an acceptablelyophilized formation.

Surprisingly and unexpectedly, however, the present inventors were ableto arrive at a solution that balances protein solubility, stability,cake structure and osmolality. More specifically, to generate a suitablelyophilized formation, an example r-Antidote solution includes about 45mM arginine (10-55 mM), about 2% sucrose (1-3%), and about 5% mannitol(2-8%). Further, the solution includes about 10 mM tris, and 0.01%-0.02%PS80 along with a desired amount of r-Antidote (e.g., 10 mg/mL, 15mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL or 50 mg/mL), and has a pH of about7.8.

Further, despite being known as a good solubilizing agent fortherapeutic proteins, citrate has been shown to have anticoagulatingactivities. See, e.g., Wright et al., Nephrology (Carlton). 2011May;16(4):396-402. Therefore, since the r-Antidote is intended as anantidote to anticoagulating agents (fXa inhibitors), citrate wasconsidered not suitable for use with the r-Antidote. Unexpectedly, it isdiscovered herein that citrate actually does not interfere with ther-Antidote's activity in vivo. A suitable concentration of citrate isfound to be from about 10 mM to about 25 mM, in addition to about 2%sucrose (1-3%), and about 5% mannitol (2-8%) in a solution suitable forlyophilization.

Accordingly, when the solution is lyophilized, it will form a drycomposition that includes a weight ratio of L-arginineHCl:sucrose:mannitol in the range of (0.5-1.4):(1-3):(2-8). If between 5mg/mL and 50 mg/mL r-Antidote is used in the solution, for instance,then the weight ratio of L-arginine HCl:sucrose:mannitol:r-Antidote inthe range of (0.5-1.4):(1-3):(2-8):(0.5-5).

Conversely, when such a lyophilized formulation is dissolved in water,saline, or other similar solvent, it can provide a solution that hasabout 10-55 mM arginine, about 1-3% sucrose, and about 2-8% mannitol.

Likewise, when a solution using citrate as the solubilizing agent islyophilized, it will form a dry composition that includes a weight ratioof citrate:sucrose:mannitol in the range of (0.15-0.66):(1-3):(2-8). Ifbetween 5 mg/mL and 50 mg/mL r-Antidote is used in the solution, forinstance, then the weight ratio of L-arginineHCl:sucrose:mannitol:r-Antidote in the range of(0.15-0.66):(1-3):(2-8):(0.5-5). Conversely, when such a lyophilizedformulation is dissolved in water, saline, or other similar solvent, itcan provide a solution that has about 8-35 mM citrate, about 1-3%sucrose, and about 2-8% mannitol.

The results observed with the r-Antidote can be readily extrapolated toother fXa antidotes that have similar structures including biologicalequivalents of r-Antidote (or their precursors, represented by SEQ IDNO: 2). In one aspect, such biological equivalents have at least 80%,85%, 90%, or 95% sequence identity to SEQ ID NO: 3. In one aspect, suchbiological equivalents include two peptide chains, each having at least80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5,respectively. In one aspect, such biological equivalents retain thestructural characteristics of SEQ ID NO: 3, that is, a modified activesite and a deleted or modified Gla domain. In another aspect, suchbiological equivalents retain the functional features of SEQ ID NO: 3,that is, not competing with fXa in assembling into the prothrombinasecomplex and having reduced or no procoagulant (e.g., enzymatic orcatalytic) activities.

Also, it is contemplated that arginine can be substituted with anothersolubilizing agent, mannitol can be substituted with anothercrystallizing agent, and sucrose can be substituted with anotherstabilizing agent, adequate examples of each of which are available inthe art and are provided in the present disclosure.

A. Polypeptide Solution Suitable for Lyophilization

In one embodiment, the present disclosure provides an aqueousformulation suitable for lyophilization, which formulation includes afXa antidote as disclosed here or its biological equivalents, along witha solubilizing agent, a stabilizing agent (or stabilizer), and acrystalline agent. The formulation can further include a surfactantand/or a buffer. In some aspects, the presence of each of these agentsprevents the fXa antidote from collapsing during lyophilization, forinstance, when the freeze-dry temperature is higher than −40° C., −30°C., −20° C., −10° C., 0° C., 5° C., 10° C., or 15° C., as high as 20° C.or 25° C.

One embodiment of the disclosure provides an aqueous formulation whichcan be used for lyophilization. The aqueous formulation includes a fXaderivative polypeptide, e.g., a polypeptide comprising the amino acidsequence of SEQ ID NO. 3 or an amino acid sequence having at least 95%sequence identity to SEQ ID NO. 3. In addition to the polypeptide, theformulation further includes a solubilizing agent, a stabilizer, and acrystalline component. Such a formulation does not collapse duringlyophilization under desired conditions. In one aspect, the desiredcondition is freeze drying at a temperature that is higher than −40° C.,or alternatively higher than −40° C., −30° C., −2° C., −10° C., 0° C.,5° C., 10° C., or 15° C. In another aspect, the desired condition isfreeze drying at a temperature that is lower than 25° C., oralternatively lower than 20° C., 15° C., 10° C., or 5° C.

In one aspect, the fXa derivative polypeptide has modifications to theGla domain and the active site as compared to the wild-type fXa protein.In one aspect, the fXa derivative polypeptide retains fXa's ability tobind to a fXa inhibitor but does not assemble into a prothrombinasecomplex. In one aspect, the fXa derivative polypeptide is a two-chainpolypeptide having an amino acid sequence of SEQ ID NO. 3, whichincludes a light chain (SEQ ID NO. 4) and a heavy chain (SEQ ID NO. 5)connected with a single disulfide bond between Cysteine 98 (Cys98) ofthe light chain and Cysteine 108 (Cys108) of the heavy chain. In oneaspect, the aqueous formulation includes at least 5 mg/mL of thepolypeptide. In one aspect, the aqueous formulation includes at least 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/mL of the polypeptide.

In some aspects, a crystalline component is included in the formulationat a concentration suitable for forming a crystalline matrix during thefreeze drying process. The formulation of the crystalline matrix isuseful for preventing collapse, as demonstrated in the examples.

A “crystalline component” refers to a molecule that forms a crystallinematrix in a formulation that includes a polypeptide, during a freezedrying process. Non-limiting examples of crystalline components includemannitol and glycine.

In some aspects, the crystalline component is mannitol (e.g.,crystalline mannitol). In one aspect, the concentration of thecrystalline component in the aqueous formulation is at least 1% (w/v).In one aspect, the concentration of the crystalline component in theaqueous formulation is at least 1.5%, 2%, 2.5%, 3%, 3.5% or 4% (w/v). Inone aspect, the concentration of the crystalline component in theaqueous formulation is not higher than 8%, or alternatively not higherthan 7%, 6.5%, 6%, 5.5%, 5%, 4.5% or 4% (w/v). In one aspect, theconcentration of the crystalline component in the aqueous formulation isfrom about 1% to about 8%, or from about 2% to about 6%, or from about3% to about 5.5%, or from about 4.5% to about 5.5%, or from about 4.6%to about 5.4%, or from about 4.7% to about 5.3%, or from about 4.8% toabout 5.2%, or from about 4.9% to about 5.1%, or at about 4%, 4.5%, or5% (w/v).

In some aspects, a solubilizing agent is included in the aqueousformulation. The term “solubilizing agent” refers to salts, ions,carbohydrates, complexation agent, polymers and other compounds which,when present in solution, increases the solubility of another molecule(e.g., an active ingredient) in the solution. Non-limiting examples ofsolubilizing agents include arginine and citrate. In one aspect, thesolubilizing agent is arginine. In one aspect, the solubilizing agent iscitrate.

The presence of the solubilizing agent is demonstrated herein to beuseful in keeping the fXa polypeptide soluble and stable in theformulation. In some aspects, the concentration of the solubilizingagent (e.g., arginine) is at least 10 mM, or alternatively at least 20mM, 25 mM, 30 mM, 36 mM, or 40 mM. In some aspects, the concentration ofthe solubilizing agent (e.g., arginine) is not higher than 100 mM, 96mM, 90 mM, 80 mM, 70 mM, 60 mM or 50 mM. In some aspects, theconcentration of the solubilizing agent is from about 10 mM or 20 mM toabout 60 mM, from about 10 mM or 20 mM to about 55 mM, from about 35 mMto about 55 mM, from about 40 mM to about 50 mM, from about 41 mM toabout 49 mM, from about 42 mM to about 48 mM, from about 43 mM to about47 mM, from about 44 mM to about 46 mM, or at about 40 mM, 45 mM or 50mM. It is noted that as used herein, the term arginine refers to theamino acid as well as the salts (e.g., arginine HCl) thereof. Argininehas a molecular weight of about 174.2 Dalton and arginine HCl (e.g.,L-arginine HCl) has a molecular weight of about 210.7 Dalton.

In one embodiment, the solubilizing agent is citrate or a salt thereof.The salt of citrate is sodium citrate. In one aspect, the citratecomprises a concentration from about 1.0 mM to about 200.0 mM. In afurther aspect, the concentration of the citrate is about 25 mM. Inanother aspect, the concentration of the citrate is about 50 mM. Infurther embodiment, the concentration of the citrate is about 5 mM, 10mM, or 20 mM. In another embodiment, the citrate comprises aconcentration from about 0.05 M to about 0.2 M.

In some aspects, a stabilizer is included in the aqueous formulation.The term “stabilizer” denotes a pharmaceutical acceptable excipient,which protects the active ingredient (e.g., the fXa derivativepolypeptides) and/or the formulation from chemical and/or physicaldegradation during manufacturing, storage and application. Examples ofstabilizers may be include sucrose, arginine, citrate, mannitol,trehalose, glycine, sodium chloride, dextran and glucose. In one aspect,the stabilizer is sucrose.

In one aspect, the concentration of the stabilizer in the aqueousformulation (e.g., sucrose) is at least about 0.5% (w/v). In one aspect,the concentration of the stabilizer in the aqueous formulation (e.g.,sucrose) is at least about 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% (w/v). In one aspect, theconcentration of the stabilizer in the aqueous formulation (e.g.,sucrose) is not greater than about 5%, 4.5%, 4%, 3.5%, 3%, 2.5% or 2%(w/v). In one aspect, the concentration of the stabilizer in the aqueousformulation (e.g., sucrose) is from about 1% to about 5%, or from about1% to about 4%, or from about 1% to about 3%, or from about 1.5% toabout 2.5%, or from about 1.6% to about 2.4%, or from about 1.7% toabout 2.3%, or from about 1.7% to about 2.2%, or from about 1.9% toabout 2.1%, or at about 1%, 1.5%, 2%, 2.5% or 3% (w/v).

In some aspects, the aqueous formulation can further include asurfactant, a buffer, a tonicity agent, a cryoprotectant, a surfactant,a lyoprotectant, a preservative or combinations thereof.

In some aspects, the aqueous formulation has a pH that is 6 or higher,or 6.5 or higher, or 7 or higher, or 7.5 or higher. In some aspects, thepH is not higher than 9, 8.5, or 8. In some aspects, the pH is between 6and 9, between 6.5 and 8.5, between 7 and 8.5, between 7.5 and 8.2,between 7.6 and 8.1, between 7.7 and 7.9, or at about 7.5, 7.6, 7.7,7.8, 7.9 or 8.

In one aspect, the aqueous formulation includes about 45 mM arginine,about 2% sucrose (w/v), about 5% mannitol (w/v) and about 10 mg/mL of atwo-chain r-Antidote, wherein the formulation has a pH of about 7.8. Inone aspect, the aqueous formulation includes about 45 mM arginine, about2% sucrose (w/v), about 5% mannitol (w/v) and about 20 mg/mL of atwo-chain r-Antidote, wherein the formulation has a pH of about 7.8. Inone aspect, the aqueous formulation includes about 45 mM arginine, about2% sucrose (w/v), about 5% mannitol (w/v) and about 40 mg/mL of atwo-chain r-Antidote, wherein the formulation has a pH of about 7.8. Inone aspect, the aqueous formulation further includes 0.01%-0.02% (w/v)Polysorbate 80 and a buffer.

B. Lyophilization and Lyophilized Compositions

Also provided, in some embodiments, are methods of lyophilizing theaqueous formulations of the present disclosure. In one aspect, thedisclosure provides a conservative lyophilization cycle as exemplifiedin Table 8.2, which includes a freezing step, an isothermal step, anannealing step, a primary drying step and a secondary drying step.

In another aspect, the lyophilization cycle includes the steps asdescribed in Table 6. It is further noted that, once an aqueous solutionsuitable for lyophilization is identified, the method of lyophilizingthe solution can be derived accordingly, with methods known in the art.In one aspect, one, or more or all of the drying steps are carried outat a temperature of −40° C. or higher. In one aspect, the drying stepsare carried out at a temperature of −35° C., −30° C., −25° C., −20° C.,−10° C. or 0° C. or higher, but not higher than 10° C., 15° C., 20° C.or 25° C.

In some aspects, also provided are lyophilized compositions prepared bylyophilizing the aqueous formulation of the present disclosure. Based onthe concentrations of each agent in the aqueous formulation, therelative content of the agent in the lyophilized composition can readilybe determined.

In one aspect, the lyophilized composition includes at least 5%, oralternatively at least 10%, 15%, 20%, 25%, 30%, or 35% (w/w) of the fXaderivative polypeptide. Then, among the other main ingredients, forinstance, there can be a weight ratio for L-arginineHCl:sucrose:mannitol in the range of (0.5-1.4):(1-3):(2-6). In someaspects, the weight ratio of L-arginine HCl:sucrose:mannitol is in therange of (0.9-1):(1.5-2.5):(4.5-5.5), or(0.91-0.99):(1.6-2.4):(4.6-5.4), or (0.92-0.98):(1.7-2.3):(4.7-5.3),(0.93-0.97):(1.8-2.2):(4.8-5.2), or (0.94-0.96):(1.9-2.1):(4.9-5.1). Insome aspects, the lyophilized composition further includes a surfactantand/or the solid portion of a buffer.

Still, in some aspects, provided is a solution prepared by dissolvingthe lyophilized composition of the present disclosure in a solvent. Insome aspects, the solvent is water or saline. In one aspect, the solventis water. In one aspect, the solution includes at least 5 mg/ml oralternatively at least 10 mg/ml of the target polypeptide.

In one embodiment, the present disclosure provides a lyophilizedcomposition comprising at least 10% (w/w) of the r-Antidote, andL-arginine HCl:sucrose:mannitol in a weight ratio of about 0.95:2:5. Inone embodiment, the present disclosure provides a lyophilizedcomposition comprising at least 20% (w/w) of the r-Antidote, andL-arginine HCl:sucrose:mannitol in a weight ratio of about 0.95:2:5. Inone embodiment, the present disclosure provides a lyophilizedcomposition comprising at least 40% (w/w) of the r-Antidote, andL-arginine HCl:sucrose:mannitol in a weight ratio of about 0.95:2:5.

III. Methods of Using the Formulations

The present disclosure also relates to therapeutic methods of treating,preventing or reducing bleeding in a subject undergoing anticoagulanttherapy with a fXa inhibitor comprising administering to a subject aneffective amount of the lyophilized formulation upon being dissolved ina suitable solvent. It is contemplated that the antidotes or derivativesof the present disclosure may be short-duration drugs to be used inelective or emergency situations, which can safely and specificallyneutralize a fXa inhibitor's conventional anticoagulant propertieswithout causing deleterious hemodynamic side-effects or exacerbation ofthe proliferative vascular response to injury.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder.

“Treating” also covers any treatment of a disorder in a mammal, andincludes: (a) preventing a disorder from occurring in a subject that maybe predisposed to a disorder, but may have not yet been diagnosed ashaving it, e.g., prevent bleeding in a patient with anticoagulantoverdose; (b) inhibiting a disorder, i.e., arresting its development,e.g., inhibiting bleeding; or (c) relieving or ameliorating thedisorder, e.g., reducing bleeding.

As used herein, to “treat” further includes systemic amelioration of thesymptoms associated with the pathology and/or a delay in onset ofsymptoms. Clinical and sub-clinical evidence of “treatment” will varywith the pathology, the individual and the treatment.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art. A “subject” ofdiagnosis or treatment is a cell or a mammal, including a human.Non-human animals subject to diagnosis or treatment include, forexample, murine, such as rats, mice, canine, such as dogs, leporids,such as rabbits, livestock, sport animals, and pets.

The agents and compositions of the present disclosure can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

An agent of the present disclosure can be administered for therapy byany suitable route, specifically by parental (including subcutaneous,intramuscular, intravenous and intradermal) administration. It will alsobe appreciated that the preferred route will vary with the condition andage of the recipient, and the disease being treated.

The phrase “pharmaceutically acceptable polymer” refers to the group ofcompounds which can be conjugated to one or more polypeptides describedhere. It is contemplated that the conjugation of a polymer to thepolypeptide is capable of extending the half-life of the polypeptide invivo and in vitro. Non-limiting examples include polyethylene glycols,polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives,polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.

“Anticoagulant agents” or “anticoagulants” are agents that inhibit bloodclot formation. Examples of anticoagulant agents include, but are notlimited to, specific inhibitors of thrombin, factor IXa, factor Xa,factor XIa, factor XIIa or factor VIIa, heparin and derivatives, vitaminK antagonists, and anti-tissue factor antibodies. Examples of specificinhibitors of thrombin include hirudin, bivalirudin (Angiomax®),argatroban and lepirudin (Refludan®). Examples of heparin andderivatives include unfractionated heparin (UFH), low molecular weightheparin (LMWH), such as enoxaparin (Lovenox®), dalteparin (Fragmin®),and danaparoid (Orgaran®); and synthetic pentasaccharide, such asfondaparinux (Arixtra®). Examples of vitamin K antagonists includewarfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®),clorindione, dicumarol, diphenadione, ethyl biscoumacetate,phenprocoumon, phenindione, and tioclomarol. In one embodiment, theanticoagulant is an inhibitor of factor Xa. In one embodiment, theanticoagulant is betrixaban.

“Anticoagulant therapy” refers to a therapeutic regime that isadministered to a patient to prevent undesired blood clots orthrombosis. An anticoagulant therapy comprises administering one or acombination of two or more anticoagulant agents or other agents at adosage and schedule suitable for treating or preventing the undesiredblood clots or thrombosis in the patient.

The term “factor Xa inhibitors” or “inhibitors of factor Xa” refer tocompounds that can inhibit, either directly or indirectly, thecoagulation factor Xa's activity of catalyzing conversion of prothrombinto thrombin in vitro and/or in vivo.

“Direct factor Xa inhibitors” bind to the fXa directly and non-limitingexamples include NAP-5, rNAPc2, tissue factor pathway inhibitor (TFPI),DX- DX-9065a (as described in, e.g., Herbert, J.M., et al, J PharmacolExp Ther. 1996 276(3):1030-8), YM-60828 (as described in, e.g.,Taniuchi, Y., et al, Thromb Haemost. 1998 79(3):543-8), YM-150 (asdescribed in, e.g., Eriksson, B.I. et. al, Blood 2005;106(11), Abstract1865), apixaban, rivaroxaban, TAK-442, PD-348292 (as described in, e.g.,Pipeline Insight: Antithrombotics—Reaching the Untreated ProphylaxisMarket, 2007), otamixaban, edoxaban (as described in, e.g., Hylek EM,Curr Opin Invest Drugs 2007 8(9):778-783), LY517717 (as described in,e.g., Agnelli, G., et al, J. Thromb. Haemost. 2007 5(4):746-53),GSK913893, razaxaban, betrixaban or a pharmaceutically acceptable saltthereof, and combinations thereof. In a particular aspect, the directfactor Xa inhibitor is rivaroxaban. In some aspects, a direct fXainhibitor is a small molecule chemical compound.

“Indirect factor Xa inhibitors'” inhibition of the fXa activity ismediated by one or more other factors. Non-limiting examples of indirectfactor Xa inhibitors include fondaparinux, idraparinux, biotinylatedidraparinux, enoxaparin, fragmin, tinzaparin, low molecular weightheparin (“LMWH”), and combinations thereof. In a particular aspect, theindirect factor Xa inhibitor is enoxaparin.

In one embodiment, the factor Xa inhibitor is selected from betrixaban,rivaroxaban, LMWH, DX-9065a, YM-60828, YM-150, PD-348292, otamixaban,edoxaban, LY517717, GSK913893, razaxaban, apixaban, and combinationsthereof.

The term “betrixaban” refers to the compound“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide”or pharmaceutically acceptable salts thereof.“2-({4[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide”refers to the compound having the following structure:

or a tautomer or pharmaceutically acceptable salt thereof.

Betrixaban is described in U.S. Pat. Nos. 6,376,515 and 6,835,739 andU.S. Patent Application Publication No. 2007/0112039, filed on Nov. 7,2006, the contents of which are incorporated herein by reference.Betrixaban is known to be a specific inhibitor of factor Xa.

“Neutralize,” “reverse” or “counteract” the activity of an inhibitor offXa or similar phrases refer to inhibit or block the factor Xainhibitory or anticoagulant function of a fXa inhibitor. Such phrasesrefer to partial inhibition or blocking of the function, as well as toinhibiting or blocking most or all of fXa inhibitor activity, in vitroand/or in vivo.

“An effective amount” refers to the amount of derivative sufficient toinduce a desired biological and/or therapeutic result. That result canbe alleviation of the signs, symptoms, or causes of a disease, or anyother desired alteration of a biological system. In the presentdisclosure, the result will typically involve one or more of thefollowing: neutralization of a fXa inhibitor that has been administeredto a patient, reversal of the anticoagulant activity of the fXainhibitor, removal of the fXa inhibitor from the plasma, restoration ofhemostasis, and reduction or cessation of bleeding. The effective amountwill vary depending upon the specific antidote agent used, the specificfXa inhibitor the subject has been administered, the dosing regimen ofthe fXa inhibitor, timing of administration of the antidote, the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, all of which can be determined readily by one of ordinaryskill in the art.

In certain aspects, the solution is administered to deliver an amount ofthe fXa derivative (e.g., the r-antidote) from about 10 milligrams (mg)to about 2 grams (g). Other amounts of the r-antidote used include fromabout 100 mg to about 1.5 g; from about 200 mg to about 1 g; and fromabout 400 mg to about 900 mg. In some aspects, the amount of ther-antidote used is about 400 mg or 960 mg. In some aspects, the amountof the r-antidote used is from about 10 mg to about 100 mg; from about15 mg to about 95 mg; and from about 20 mg to about 80 mg.

In another embodiment, the solution administered in a neutralizingamount that is at least about a 1:1 fold molar ratio of circulatingconcentration of r-antidote over circulating concentration of the factorXa inhibitor for a period of at least about 30 minutes. In otherembodiments the molar ratio is about 1:1 or about 2:1 or about 4:1.

The formulation when administered neutralizes the factor Xa inhibitor byat least about 20%, or by at least about 50%, or by at least about 75%,or by at least about 90%, or by at least about 95%.

One can determine if the method, i.e., inhibition or reversal of afactor Xa inhibitor is achieved, by a number of in vitro assays, such asthrombin generation assay, and clinical clotting assays such as aPTT, PTand ACT.

One aspect of the present disclosure relates methods of selectivelybinding and inhibiting an exogenously administered fXa inhibitor in asubject undergoing anticoagulant therapy with a fXa inhibitor comprisingadministering to the subject an effective amount of a solution of thelyophilized formulation. Patients suitable for this therapy haveundergone prior anticoagulant therapy, for example, they have beenadministered one, or more of an anticoagulant, such as a direct orindirect inhibitor of fXa.

In some embodiments, the solution is administered after theadministration of an overdose of a fXa inhibitor or prior to a surgery,which may expose subjects to the risk of hemorrhage. The subject may bea cell or a mammal, such as a human.

In another aspect the method provide herein selectively binds andinhibits an exogenously administered factor Xa inhibitor in a subjectundergoing anticoagulant therapy with a factor Xa inhibitor comprisingadministering a solution of the lyophilized formulation to the subject.The subject may be a cell or a mammal, such as a human.

Subjects that will benefit from the administration of the dissolvedlyophilized formulation described herein and the accompanying methodsinclude those that are experiencing, or predisposed to a clinical majorbleeding event or a clinically significant non-major bleeding event.Examples of clinical major bleeding events are selected from the groupconsisting of hemorrhage, bleeding into vital organs, bleeding requiringre-operation or a new therapeutic procedure, and a bleeding indexof >2.0 with an associated overt bleed. (Turpie AGG, et al, NEJM, 2001,344: 619-625.) Additionally, the subject may be experiencing orpredisposed to a non-major bleeding event selected from the groupconsisting of epistaxis that is persistent or recurrent and insubstantial amount or will not stop without intervention, rectal orurinary tract bleeding that does not rise to a level requiring atherapeutic procedure, substantial hematomas at injection sites orelsewhere that are spontaneous or occur with trivial trauma, substantialblood loss more than usually associated with a surgical procedure thatdoes not require drainage, and bleeding requiring unplanned transfusion.

In some embodiments, the dissolved lyophilized formulation isadministered after the administration of an overdose of a fXa inhibitoror prior to a surgery, which may expose subjects to the risk ofhemorrhage.

In any of the methods described herein, it should be understood, even ifnot always explicitly stated, that an effective amount of the dissolvedlyophilized formulation is administered to the subject. The amount canbe empirically determined by the treating physician and will vary withthe age, gender, weight and health of the subject. Additional factors tobe considered by the treating physician include but are not limited tothe identity and/or amount of factor Xa inhibitor, which may have beenadministered, the method or mode that the lyophilized formulation willbe administered to the subject, and the therapeutic end point for thepatient. With these variables in mind, one of skill will administer atherapeutically effective amount to the subject to be treated.

EXAMPLES

The disclosure is further understood by reference to the followingexamples, which are intended to be purely exemplary of the disclosure.The present disclosure is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe disclosure only. Any methods that are functionally equivalent arewithin the scope of the disclosure. Various modifications of thedisclosure in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

-   -   hr=hour    -   INR=international normalized ratio    -   IV=intravenous    -   kg=kilogram    -   M=molar    -   mg=milligram    -   mg/kg=milligram/kilogram    -   mg/mL=milligram/milliliter    -   min=minute    -   mL=milliliter    -   PPP=platelet poor plasma    -   PRP=platelet rich plasma    -   PT=prothrombin time    -   U/mL=units/milliliter    -   μL or uL=microliter    -   μM=Micromolar

Example 1. Preparation of r-Antidote

Citrate-phosphate (20 mM) buffers with an ionic strength of 0.15(adjusted with NaCl) were prepared using citric acid monohydrate(Fisher, Pittsburgh, Pa.) and sodium phosphate dibasic, anhydrous(Sigma, St. Louis, Mo.) and the pH was adjusted using either 6 M HCl or6 M NaOH. Phosphate (20 mM) buffer without additional salt was preparedby dissolving 6.61 g of sodium phosphate dibasic anhydrous in 2.0 L ofMili-Q water and pH adjusted to 7.5. For the phosphate (20 mM) buffercontaining salt (I=0.15 M), 14.8 g NaCl was added to the phosphatebuffer described above. All other reagents were purchased from Sigma(St. Louis, Mo.) unless otherwise noted.

The polypeptide of the r-Antidote (SEQ ID NO. 3) was stored in stocksolution of a concentration of approximately 5 mg/ml in 10 mM Tris, pH8.0 containing 2% arginine. The dialysis of the r-Antidote was performedat 4° C. using Slide-A-Lyzer® Dialysis Cassettes, 3000 MWCO (Pierce,Rockford, Ill.) against citrate-phosphate buffers of selected pH values.To prevent aggregation during dialysis, the stock protein solution wasdiluted to 0.5 mg/ml with filtered dialysis buffer prior to loading intocassettes. After dialysis, the r-Antidote was diluted to 0.3 mg/ml andprotein concentration was measured with UV absorbance spectroscopy(A₂₈₀) using an extinction coefficient of 1.16 ml·mg⁻¹·cm⁻¹. Ther-Antidote produced by this method was used in the following examples.

Example 2. Differential Scanning Calorimetry (DSC) for StabilityMonitoring

Differential Scanning Calorimetry (DSC) was performed using a Microcalcapillary auto-DSC with a temperature controlled sample loading chamber.Thermal ramps were performed from 6-100° C. with a scanning rate of 60°C./hr and a 25 minute pre-scanning equilibration period. Appropriatematching buffer was used in the reference cell, while typical sampleconcentrations in matching buffer were ˜0.6 mg/mL. A buffer versusbuffer reference scan was subtracted from all sample scans and thethermograms were concentration normalized prior to analysis. Data wereprocessed using the supplied software from Microcal. Endothermic peakswere fit to a single peak using a non two-state fitting function, andtransition temperature (Tm) values were calculated by the fittingfunction. The onset temperatures (Tonset) values were determined by thedeviation of the endothermic peak from a low temperature baseline.

DLS is often employed to show the presence of multiple populations in aheterogeneous sample. Using a Wyatt plate reader DLS instrument, 10-20μL of protein solution (0.3 mg/mL) at different pH conditions wasmeasured in a single set of experiments at 20° C. The samples werecentrifuged for 5 min at 3000 rpm to remove any air bubbles, and 5 scansof 20 seconds each were acquired to give an average sample radius. At pH5.0-7.5, a single population with a hydrodynamic radii·3 nm wereobserved.

Example 3. Identification of Stabilizers

Data from Example 2 demonstrated that the r-Antidote was overall stableat pH 7.5. Thus, the excipient screening studies were performed at pH7.5 in a 20 mM phosphate buffer. The hydrodynamic diameter of theprotein was measured using a Dynapro dynamic light scattering platereader instrument (Wyatt Technology, Santa Barbara, Calif.). Thehydrodynamic diameter was calculated from the diffusion coefficient bythe Stokes-Einstein equation using the method of cumulants (lognormalnumber based). The measurements were used to evaluate the homogeneity ofthe supplied sample.

A SpectraMax M3 plate reader was first employed to identify potentialstabilizing excipients by monitoring the protein aggregation kinetics at60° C., pH 7.5 with 0.3 mg/ml protein with or without excipients. Intotal, 32 excipients from a library of Generally-Regarded-As-Safe (GRAS)excipients were tested as listed in Table 4.1.

TABLE 4.1 Excipient List and Concentrations Tested by OD 350 nm KineticsStudy Excipient Concentration Excipient Concentration Dextran Sulfate0.0075 mM Tween 20  0.10% Dextran T70 0.0075 mM Tween 80  0.10% Ascorbicacid 0.15M Pluronic F-68  0.10% Aspartic Acid 0.15M Albumin  5.00%GlutamicAcid 0.15M Gelatin  5.00% Lactic Acid 0.15M Lactose  20.0% MalicAcid 0.15M Trehalose 10.00% Arginine 0.3M Dextrose 20.00% Diethanolamine0.3M Sucrose  20.0% Guanidine 0.3M Mannitol 10.00% Lysine 0.3M Sorbitol20.00% Proline 0.3M Glycerol 20.00% Glycine 0.3M α Cyclodextrin  2.50%Calcium Chloride 0.015M 2-OH propyl β-CD 10.00% Sodium Citrate 0.2M 2-OHpropyl γ-CD 10.00% Brij 35 0.10% EDTA 1 mM & 5 mM

It was determined that of the excipients tested, sucrose, sorbitol andcitrate had the largest stabilization effect. The subsequent testing ofthe effect of excipient combinations on protein stability were based onthese three excipients as summarized in Table 4.2. The OD 350 nm meltswere run in duplicate and the ΔT values for each formulation werecalculated as described above. Based on ΔT values shown in Table 2,formulations 3, 4, 5 and 6 were identified to have the greateststabilizing effect.

TABLE 4.2 List of Excipient Combinations Tested and the Corresponding ΔTValues and Solution Osmolality. ΔT Osmolality No. Components (° C.)(mOsm/kg) 1 Sucrose 10% + Sorbitol 5% 3.1 943 ± 13  2 Sucrose 5% +Sorbitol 10% 3.8 1066 ± 8   3 Sucrose 10% + Sorbitol 5% + >10.0 1098 ±21  Sodium Citrate 0.05M 4 Sucrose 5% + Sorbitol 10% + >10.0 1253 ± 8  Sodium Citrate 0.05M 5 Sucrose 5% + Sorbitol 5% + 6.2 863 ± 10  SodiumCitrate 0.05M 6 Sucrose 10% + Sodium Citrate 0.05M 6.5 755 ± 9  7Sorbitol 10% + Sodium Citrate 0.05M 5.8 1008 ± 6   The ΔT is thedifference between transition temperatures of protein alone and proteinwith different combination of excipients at pH 7.5. The osmolality isaveraged from triplicate measurements and ΔT is averaged from duplicatemeasurements.

The aggregation properties of the therapeutic protein in theseformulations were further studied using the OD 350 nm melt method in 20mM phosphate buffer at pH 7.5, without NaCl, in these combinationformulations. In general, the extent of aggregation is much lowerwithout NaCl. In fact, the OD 350 nm melt was initially run from 35-75°C., and since no obvious aggregation was observed, the melt experimentswere re-run with the same samples from 75 to 100° C. Thus, a break canbe observed in the OD 350 nm curve at 75° C. due to ˜10 min dwell atthis temperature. No obvious aggregation was observed for proteins informulations 3, 4, 5 and 6 even after ramping up to 100° C. Anotherbenefit of removing additional NaCl is that the corresponding osmolalityof the formulations is much lower compared to those formulations withNaCl.

Example 4. Solubility Testing

This example tests the effect of pH, temperature, stabilizers (e.g.,citrate, arginine, glycine, and lysine) and ionic strength on thesolubility of the r-Antidote.

Material & Methods

The material used was a solution of r-Antidote (4.8 mg/ml) in 10 mM TrispH8.0, and 2% arginine. For solubility at room temperature (RT), testingwas conducted by physical observation for at least 1-2 hrs. Forsolubility at 5° C., samples were equilibrated at 5° C. overnight andphysical observation was conducted on the samples. In addition, thesamples were centrifuged at 5° C. for 15 min and protein concentrationsin supernatant were analyzed by UV A280 nm (duplicate dilution). Theoriginal stock solution was analyzed daily as control.

When protein precipitation was observed, the solubility determined fromsupernatant concentration was interpreted as <XX mg/mL (shaded bars inthe corresponding figures). This is due to excess amount of proteinpresence and preferential precipitation of a sub-population of proteinthat has PI close to the buffer pH. When protein precipitation is notobserved, the solubility determined from solution concentration wasinterpreted as >XX mg/mL (empty bars in the figures).

Impact of pH on solubility at room temperature was tested with differentpH, including 5.0, 6.0, 7.0 and 8.0. As shown in FIG. 1A, the r-Antidotehad the highest solubility at pH 8.0 (42.2 mg/mL with no visibleprecipitation). By contrast, the solubility was 3.5 mg/mL (noprecipitation), 12.3 mg/mL (precipitation observed) and 24.4 mg/mL(precipitation observed) at pH 5.0, 6.0 and 7.0, respectively.

Table 5.1 lists samples tested for the solubility at 5° C. As shown, theUF buffer is composed of 42 mM MES, 4 mM Sodium Phosphate, 833 mM NaCl,8 mM Tris, and 58 mM arginine concentrated to ˜5 mg/mL.

TABLE 5.1 Samples tested for r-Antidote solubility at different pH withdifferent solubilizers (citrate or arginine) pH Calculated SampleComposition Citrate/Arginine (±0.02) Osmolality 10 mM Citrate 10 mM Naphos, 9.5% sucrose, 10 mM Citrate 7.30 353 0.01% PS80, 10 mM Citrate  3mM Citrate 10 mM Na phos, 9.5% sucrose,  3 mM Citrate 7.30 325 0.01%PS80, 3 mM Citrate  0 mM Citrate 10 mM Na phos, 9.5% sucrose,  0 mMCitrate 7.30 313 0.01% PS80 pH 7.80 10 mM Na phos, 9.5% n/a 7.80 313sucrose, 0.01% PS80 pH 7.55 10 mM Na phos, 9.5% sucrose, n/a 7.55 3130.01% PS80 UF buffer 42 mM MES, 4 mM NaPhos. 58 mM Arg 7.48 1942 833 mMNaCl, 8 mM Tris, 58 mM Arg

At pH 7.3, 10 mM citrate improved antidote 5° C. solubility slightly.Without citrate or arginine, 5° C. solubility had following rank order:pH 7.55>pH 7.80>pH 7.30 (FIG. 1B). The UF buffer (pH 7.48) appeared tohave best solubility (50 mg/mL), likely due to presence of 58 mM Arg+833mM NaCl at the suitable pH, 7.5 (FIG. 1B).

Table 5.2 lists samples for testing the effect of arginine versuscitrate at pH 7.55. As shown in FIG. 1C, at pH 7.55, both citrate andarginine improved r-Antidote 5° C. solubility significantly. Further, itappeared that Citrate was more effective than arginine at the samemolarity: −10 mM citrate˜50 mM arginine>20 mM arginine>10 mM arginine.

TABLE 5.2 Samples tested for r-Antidote solubility in citrate andarginine at pH 7.55 pH Cacl Sample Composition Citrate/Arg (±0.02) Osm 0 mM 10 mM Na phos, 9.5% sucrose, n/a 7.55 313 0.01% PS80 (set 1) 10 mM10 mM Na phos, 8% sucrose, 10 mM 7.55 307 Citrate 0.01% PS80, 10 mMCitrate Citrate 10 mM Arg 10 mM Na phos, 8% sucrose, 10 mM Arg 7.55 2970.01% PS80, 10 mM Arg 20 mM Arg 10 mM Na phos, 8% sucrose, 20 mM Arg7.55 327 0.01% PS80, 20 mM Arg 50 mM Arg 10 mM Na phos, 8% sucrose, 50mM Arg 7.55 417 0.01% PS80, 50 mM Arg

The effect of arginine versus citrate was further tested at pH 7.8 and8.0, using the samples in Table 5.3. FIG. 1D shows that the r-Antidotewas slightly more soluble at pH 8.0 than at pH 7.8 at 5° C. Both 10 mMCitrate and 20 mM Argnine improved solubility to at least 15 mg/mL at pH7.8 and 8.0.

TABLE 5.3 Samples tested for r-Antidote solubility in citrate andarginine at pH 7.8 and 8 Additional Citrate/ pH Cacl Sample BaseComposition Arg (±0.02) Osm pH 7.8 10 mM Na phos, 9.5% n/a 7.80 313sucrose, 0.01% PS80 (set 1) pH 7.8, 10 mM Na phos, 8% sucrose,  10 mM7.80 307 10 mM 0.01% PS80, 10 mM Citrate Citrate Citrate pH 7.8, 10 mMNa phos, 8% sucrose,  20 mM 7.80 327 20 mM 0.01% PS80, 20 mM Arg Arg ArgpH 8.0 10 mM Na phos, 8% n/a 8.00 267 sucrose, 0.01% PS80 pH 8.0, 10 mMNa phos, 8% sucrose,  10 mM 8.00 307 10 mM 0.01% PS80, 10 mM CitrateCitrate Citrate pH 8.0, 10 mM Na phos, 8% sucrose,  20 mM 8.00 327 20 mM0.01% PS80, 20 mM Arg Arg Arg Tris/Arg 10 mM Tris, 2% Arg 114 mM 8.00352 Arg

The effect of arginine was also compared to glycine and lysine at pH 7.8(Table 5.4) and the results shown in FIG. 1E. As shown in the figure,Glycine and Lysine did not have an effect on r-Antidote solubility at 5°C. and more solubilizing effect observed for 20 mM Arg at pH 8.0 vs. pH7.55 at 5° C.

TABLE 5.4 Samples tested for r-Antidote solubility in glycine, lysineand arginine at pH 7.8 Additional pH Cacl Sample Base CompositionGly/Lys/Arg (±0.02) Osm pH 7.80 10 mM Na phos, 9.5% sucrose, n/a 7.80313 0.01% PS80 pH 7.8, 10 mM Na phos, 8% sucrose, 20 mM Gly 7.80 307 20mM 0.01% PS80, 20 mM Gly Glycine pH 7.8, 10 mM Na phos, 8% sucrose, 20mM Lys 7.80 327 20 mM 0.01% PS80, 20 mM Lys Lysine pH 7.8, 10 mM Naphos, 8% sucrose, 20 mM Arg 7.80 327 20 mM 0.01% PS80, 20 mM Arg Arg(set 3) (set 3) pH 7.55, 10 mM Na phos, 8% sucrose, 20 mM Arg 7.55 32720 mM 0.01% PS80 Arg

The effect of ionic strength on the solubility of the r-Antidote wasalso tested (Table 5.5). As shown in FIG. 1F, ionic strength increasedr-Antidote solubility at 5° C. in the absence of Arginine or Citrate,and the effect appeared prominent at ionic strength>0.10 M.

TABLE 5.5 Samples tested for r-Antidote solubility at different ionicstrength at pH 7.8 Additional pH Cacl Sample Base CompositionGly/Lys/Arg (±0.02) Osm pH 7.8, 0.03M 10 mM phos, 8% sucrose, n/a 7.80277 IS (set 1) 0.01% Tween 80, 100 mL pH 7.8, 0.03M 10 mM phos, 8%sucrose, n/a 7.80 277 IS 0.01% Tween 80, 100 mL pH 7.8, 0.10M 10 mMphos, 8% sucrose, n/a 7.80 417 IS 0.01% Tween 80, 100 mL pH 7.8, 0.30M10 mM phos, 8% sucrose, n/a 7.80 817 IS 0.01% Tween 80, 100 mL pH 7.8,1.00M 10 mM phos, 8% sucrose, n/a 7.80 2217 IS 0.01% Tween 80, 100 mL

In summary, this example demonstrates that at room temperature in theabsence of a solubilizing agent such as arginine and citrate, ther-Antidote has the highest solubility at pH 8.0. At 5° C., pH 8.0 wasthe best to the r-Antidote. Further, both citrate and arginine improvethe r-Antidote's 5° C. solubility significantly. Glycine and lysine bothof which increase the Tm for the r-Antidote, however, has no effect onsolubility. Overall, the highest solubility of the r-Antidote at 5° C.was achieved at pH 7.8 with 95 mM Arginine. No precipitation wasobserved after 10 days.

Example 5. Initial Lyophilization Process

The lyophilization process was developed using a rational approach basedon an understanding of the physical nature of the formulation componentsat different stages of the lyophilization cycle. Thermalcharacterization methods including DSC and freeze dry microscopy (FDM)were used to measure Tg′ (glass transition temperature of the frozenconcentrate) and Tc (collapse temperature during primary drying). Thecycle shown in Table 6 was selected for lyophilization of thelyophilized formulation. The annealing step allows crystallization ofmannitol to ensure that product temperature does not fall below collapsetemperature during primary drying. The primary drying temperature wasselected to avoid cake collapse with a reasonable duration of primarydrying. The 2-step secondary drying condition was developed to produce alyophilized formulation with a moisture level of <1%.

TABLE 6 Lyophilization Cycle Step # Process Step Description 1 FreezingCooling at 1° C./min to −40° C. 2 Freezing Isothermal Hold at −40° C.for at least 180 min 3 Annealing Ramp to −20° C. at 1° C./min and holdfor at least 180 min 4 Freezing Cooling at 1° C./min to −40° C. and holdfor at least 180 min 5 Evacuation Initiate vacuum to 100 mTorr 6 PrimaryDrying Ramp 0.5° C./min to 10° C. Hold for 40 hours 7 Secondary Drying 1Ramp to 30° C. at 0.5° C./min, hold for 20 hours at 75 mTorr

Example 6. Lyophilization without a Crystallizing ComponentExperimental/Study Design

Ten different formulations were prepared to test the effects of buffercomposition, pH, stabilizer, and drug concentration on the solubilityand stability of the r-Antidote (Table 7). The formulations wereprepared using Tris or a phosphate buffer at pH 7.8 and 8.2. Solutionswere concentrated to 10 mg/mL and 25 mg/mL using centrifugal filtration.

Samples of the concentrated solutions prepared in tris or phosphatebuffers were placed on short term stability at 2-8° C. and 25° C. for 2weeks. At the same time, samples of each solution were used forfreeze/thaw studies and examined for precipitation and aggregation.Samples for freeze/thaw studies consisted of 0.5 mL of each formulationin a 2 mL, Type I, glass tubing vial. The 0.5 mL sample was visuallyinspected prior to freezing and after each freeze/thaw cycle. Eachsample was placed at −80° C. for approximately 2 hours, thawed forapproximately 15 to 30 minutes at room temperature, visually inspectedfor approximately 1-2 minutes, and returned to the −80° C. freezer. A250 L sample of each formulation was removed after the 3rd freezingcycle and submitted to the lab for assays. The remaining solution wassubjected to 2 additional freezing cycles and then submitted to the labfor assays.

All remaining solution was lyophilized as 0.25 mL samples using aconservative cycle and the samples were placed on accelerated stabilityat 25° C. and 40° C.

Two additional formulations were prepared using polysorbate-freesolution to test the effects of freeze/thaw and lyophilization on themolecule without the presence of protective agents (Formulations 5 and 6in Table 7). Samples of the solutions were reserved and used for thermalcharacterization using modulated DSC and freeze dry microscopy.

TABLE 7 Formulation Numbers, Components, Concentrations, and pH ValuesNo. Components Concentration pH 1A 10 mM Tris, 4% Sucrose, 95 mM 10mg/mL 7.8 Arginine, PS80 0.01% 1B 10 mM Tris, 4% Sucrose, 95 mM 25 mg/mL7.8 Arginine, PS80 0.01% 2A 10 mM Tris, 4% Sucrose, 95 mM 10 mg/mL 8.2Arginine, PS80 0.01% 2B 10 mM Tris, 4% Sucrose, 95 mM 25 mg/mL 8.2Arginine, PS80 0.01% 3A 10 mM Phos, 4% Sucrose, 95 mM 10 mg/mL 7.8Arginine, PS80 0.01% 3B 10 mM Phos, 4% Sucrose, 95 mM 25 mg/mL 7.8Arginine, PS80 0.01% 4A 10 mM Phos, 4% Sucrose, 95 mM 10 mg/mL 8.2Arginine, PS80 0.01% 4B 10 mM Phos, 4% Sucrose, 95 mM 25 mg/mL 8.2Arginine, PS80 0.01% 5 10 mM Tris, 95 mM Arginine 25 mg/mL 7.8 6 10 mMPhos, 95 mM Arginine 25 mg/mL 7.8The formulations were prepared using the bulk drug substance supplied at3 mg/mL, 3.3 mg/mL, and 4.8 mg/mL with and without polysorbate 80(PS80).

Formulations 5 and 6 were prepared first using 19 mL of the bulk drugsolution for each formulation. The volume of bulk was placed in adialysis cassette having a 10 K membrane and the cassette was placed in2 L of 10 mM Tris or 10 mM sodium phosphate buffer at pH 7.8. Thesolutions dialyzed for approximately 2 hours, the dialysis solution wasreplaced with another fresh 2 L of buffer solution and dialyzed for atleast another 2 hours. The solution was removed from each cassette andplaced in Amicon Ultra Ultracel 10K centrifugal filter tubes. Thesolutions were centrifuged for approximately 30 minutes at 3/4 speed.The remaining solution was removed from the centrifuge tubes and 95 mMArginine was added followed by adjusting the pH and volume of theconcentrated solution.

The same procedure was used to prepare formulations 1A through 4A and 1Bthrough 4B. Formulations prepared using a bulk solution with 3 mg/mLused 13.5 mL of bulk to prepare the 10 mg/mL solutions and 33.5 mL ofbulk to prepare the 25 mg/mL solutions. Formulations that used the 4.8mg/mL bulk solution used 8.4 mL of the bulk to prepare the 10 mg/mLsolution and 20.9 mL of bulk to prepare the 25 mg/mL solution.

The concentrations of sucrose and arginine needed for the final volumeof sample solution were added to the bulk solution after concentratingthe solution and then the solution was adjusted to the appropriate pHand final volume. PS80 was spiked into the final sample solutions tocreate a concentration of 0.01% using a 1% solution of PS80.

The solutions were filtered through a 0.22 μm syringe filter and thendivided into vials. 2 mL vials were each filled with 250 L of solutionand lyophilized using the following conditions:

-   -   1. Cool to −40° C. at 1° C./min    -   2. Hold at −40° C. for 1 hour then initiate vacuum at 100 mTorr    -   3. Ramp to −35° C. at 0.5° C./min and hold until the Pirani        gauge measurement matches the capacitance manometer measurement        of 100 mTorr and the product temperature reaches the shelf        temperature.    -   4. Ramp to 20° C. at 0.5° C./min and hold until the Pirani gauge        measurement matches the capacitance manometer measurement of 100        mTorr and the product temperature reaches the shelf temperature.

Stoppers were seated and vials were capped after lyophilization. Sampleswere submitted for initial time point (T0) testing and the remainingvials were placed on stability.

Modulated Differential Scanning Calorimetry (DSC)

The thermal behavior of solution samples was examined using modulatedand standard DSC. Samples were examined by placing 12 L of solution intoTzero pans and hermetically sealed. The solutions were cooled to −40° C.at 1° C./min and held isothermally for 5 minutes. The temperature of thesamples was ramped to 10° C. at 0.5° C./min with a modulation of 1° C.every 120 seconds. Some samples were examined using an annealing step.Those samples were examined by cooling to −40° C. at 1° C./min, holdingisothermally for 5 min, ramping the temperature to −15° C. or −20° C. at1 to 5° C./min and holding isothermally for at least 60 minutes. Thetemperature of the samples was returned to −40° C. at 5° C./min, heldisothermally for 5 min, and ramped at 0.5° C./min with a modulation of1° C. every 120 seconds.

Freeze-Dry Microscopy

Samples were examined using freeze-dry microscopy by placing 2 to 4 L ofsolution between 2 glass coverslips in a Linkam freeze-dry microscopestage. The sample was cooled to −40° C. or lower at 1° C./min and heldisothermally for 2 min. Vacuum was initiated at 100 micron and thesample was visually examined using a video camera mounted on a polarizedlight microscope. The sample was freeze dried at that temperature untildried material was visible and photographed. Afterward, the temperatureof the sample was increased in 2° C. increments and held at eachtemperature to observe the freeze dried sample. The temperature of thesample was increased until complete collapse was observed.

Analytical Methods A. Concentration by UV-Vis

Concentration of the solutions were measured using a Nano Drop 2000spectrophotometer (Thermo Scientific). Scans were conducted by placing 2L of solution on the testing platform and scanning in the range of 280nm.

B. pH

The pH of solutions was measured using an Orion pH meter model 920A. Themeter/probe was calibrated in the range of pH 7 to pH 10 using pre-madebuffer solutions purchased from Thermo Scientific.

C. SEC-HPLC

Size Exclusion HPLC analysis was performed using an Agilent 1100 SeriesHPLC. A mobile phase prepared at 0.1 M Sodium Phosphate, 0.75 M ArginineHydrochloride at pH 7.4 was used for separation. The analytical columnused was an YMC-Pack diol-200, 300×4.6 mm, 5 μm average particle size.The suitability of the HPLC system, including the column, was verifiedusing six replicate injections of reference material and evaluated forretention time, area, and percent area for the main protein peak.Additionally, a gel filtration standard was used to evaluate theseparation capability of the column. Samples were diluted to 1 mg/mLprotein using the formulation buffer and injected to obtain a columnload of 50 μg of protein per injection.

D. RP-HPLC

Reversed Phase HPLC analysis was performed using an Agilent 1100 SeriesHPLC. The method employs a gradient for separation using mobile phasesprepared at 0.1% Trifluoroacetic Acid in HPLC grade water and 0.08%Trifluoroacetic Acid in Acetonitrile. The analytical column used was aVydac C18 column, 150×4.6 mm, 5 μm average particle size. Thesuitability of the HPLC system including the column was verified usingsix replicate injections of reference material and evaluation ofretention time, area, and percent area for the main protein peak.Samples were diluted to 1 mg/mL protein using the formulation buffer andinjected to obtain a column load of 25 μg of protein/injection.

E. IEX

Ion Exchange HPLC analysis was performed using an Agilent 1100 SeriesHPLC. The method employs a gradient using mobile phases prepared at 20mM Sodium Phosphate at pH 6.5 and 20 mM Sodium Phosphate, 1 M SodiumChloride at pH 6.5. The analytical column used was a Dionex PropacWCX-10, 250×4 mm. The suitability of the HPLC system including thecolumn was verified using six replicate injections of reference materialand evaluation of retention time, area, and percent area for the peaklabeled as peak #2. Samples were diluted to 1 mg/mL protein using theformulation buffer and injected to obtain a column load of 50 μg ofprotein per injection.

Results

A subset of the solution samples were lyophilized using a conservativecycle and placed on stability for 2 months at 25° C. and 40° C. Thelyophilization cycle was completed within approximately 20 hours due tothe low fill volume. All lyophilized cakes appeared acceptable exceptfor formulation 2A possibly due to a filter tear.

In general, data obtained using SEC and RP appeared to distinguishdifferences between the formulations. This suggests that the methods arestability indicating and can be used for comparing samples. The datasupport that the stability of the r-Antidote is affected by pH. The datademonstrate that the stability of formulations prepared at pH 7.8 isbetter than the stability of formulations prepared at pH 8.2. This isespecially true for the samples stored at 40° C.

This study included a comparison of the buffer type on the stability ofthe r-Antidote. The buffers included tris and phosphate prepared at pH7.8 and 8.2. The data suggest that buffer type did not affect thestability of the r-Antidote and that differences in stability weremainly a function of pH.

Two formulations in the study (formulations 5 and 6) were preparedwithout sucrose and polysorbate 80. Sucrose is used as a lyoprotectantand polysorbate 80 is used to prevent aggregation of proteins due tointeractions with the walls of the vial and interactions with ice duringthe freezing step. The formulations prepared without the protectantsexhibited increases in percent aggregates as determined by SEC after 1month of storage at 40° C. The data support the need for the excipientsin the formulations to improve the stability of the protein.

The stability study also supports that the lyophilized samples are morestable than the formulations prepared as solutions. Comparison of thesolution samples demonstrates that the stability of the solution samplesis better when stored at 5° C. than when stored at higher temperatures.

The samples for the stability study were prepared using 0.25 mL per 2 mLvial. Collapse was only observed when there were insufficient solidspresent to support a cake in sample 2A. All other samples appearedacceptable, however it was not feasible to determine the extent of cakeshrinkage when using such low fill volumes. Thermal characterizationstudies were conducted concurrently with the stability studies todetermine the feasibility of lyophilizing the formulations at fullscale.

Formulations 5 and 6 were examined using modulated DSC. Bothformulations contain approximately 25 mg/mL the r-Antidote and 95 mMarginine HCl, but formulation 5 was prepared with 10 mM Tris andformulation 6 was prepared with 10 mM phosphate. No thermal events wereobserved during the warming ramp when observed using total heat flow,non-reversing heat flow, or reversing heat flow. A total heat flowthermogram will show both kinetically related events and non-kineticallyrelated events. Non-reversing heat flow thermograms will showkinetically related events such as crystallization and reversing heatflow thermograms will show non-kinetically related events such as glasstransitions. The lack of observable events may suggest that theconcentrations of the components are too low to produce a signal withsufficient intensity.

Freeze dry microscopy experiments were conducted to determine if thecollapse temperature would match the results observed for the Tg′determined using MDSC. The thermal behavior of the all of the sampleswas expected to be similar because all had the same excipients atsimilar concentrations.

Formulations 5 and 6 were prepared with a r-Antidote concentration ofapproximately 25 mg/mL and both contained 95 mM arginine at pH 7.8. Theonly difference between the formulations was the buffer. Formulation 5contained 10 mM tris and formulation 6 contained 10 mM phosphate.Formulation 5 exhibited collapse at −40° C. and formulation 6 exhibitedcollapse at −39° C. The data support that the temperature of the productneed be maintained below the determined collapse temperature to obtainacceptable lyophilized samples. Maintaining such low producttemperatures is not feasible in laboratory or full scale lyophilizers.

Although the stability data for the lyophilized formulations appearedacceptable, the thermal characterization data demonstrated that theformulation was not amenable to scale-up due to the low collapsetemperature. Thermal characterization obtained using MDSC and freeze drymicroscopy suggest that the formulations remain amorphous after freezingand drying and that the combination of components leads to a lowcollapse temperature. The only way to create a formulation that isamenable to scale up is to add a crystallizing component to serve as ascaffold that can hold the amorphous material in place during and afterfreeze drying. The most common crystallizing component added topharmaceutical formulations is mannitol. All further formulation andprocess development work investigated the addition of mannitol atdifferent concentrations. The development of a formulation containingmannitol and the stability studies for the formulations are described ina separate development report.

Conclusion

The effects of buffer type, pH, stabilizer, and protein concentration onthe stability of the r-Antidote were examined as solution andlyophilized formulations. Solution samples were stored at 5° C. and 25°C. for up to 2 weeks and lyophilized samples were stored at 25° C. and40° C. for up to 2 months. Formulations lyophilized as 0.25 mL in 2 mLvials exhibited acceptable stability after 2 months. However, thermalcharacterization experiments demonstrated that all formulations hadcollapse temperatures of −37° C. or lower and were not amenable toscale-up. The data suggest that a crystallizing component is needed inthe formulation to prevent collapse and to allow for lyophilizing athigher temperatures.

Example 7. Effect of Buffer Type and Mannitol on the Thermal Behaviorand Stability of the Formulation

Data from Example 6 suggested that a crystallizing component was neededin the r-Antidote formulation to prevent collapse during freeze drying.This example examined the effects of mannitol and arginineconcentrations on the thermal behavior and lyophilized cake appearanceof the formulations. Formulations containing 2% to 4% mannitol wereinvestigated along with reducing the concentration of arginine. Arginineprevented the crystallization of mannitol unless the concentration was47.5 mM or less. Studies found that a formulation containing 10 mM tris,10 mg/mL r-Antidote, 45 mM arginine, 2% sucrose, 5% mannitol, and 0.01%polysorbate 80 resulted in lyophilized cakes with acceptable appearance,and physical and chemical stability. Lyophilization studies provideddata to support using a primary drying shelf temperature of −25° C.after annealing at −25° C. for 3 hours. A two-step secondary dryingprocess results in cakes with residual moisture values less than 1%.

Experimental/Study Design

Studies were designed to concurrently examine the thermal behavior ofthe formulations and compare the chemical stability of formulations thatwere lyophilized using a conservative cycle. The initial formulationswere prepared with 95 mM arginine, 2% sucrose, 2% mannitol, and either10 mM tris or 10 mM phosphate buffers at pH 7.8. The formulations alsocontained the active ingredient at either 10 or 25 mg/mL (Table 8.1).

Aliquots of the thawed drug solution were placed in dialysis cassetteswith 3K molecular weight cut off (MWCO) membranes. The cassettes wereplaced in the buffer solutions containing either tris or phosphate witharginine, sucrose, and mannitol. Each cassette containing the drugsolution was placed in 2 L of buffer solution and dialyzed for 4 hours.The buffer solution was refreshed after 2 hours and the solutions weredialyzed for another 4 hours or overnight at 2-8° C. The solutions wereremoved from the dialysis cassettes using BD syringes with 18G needlesand placed in centrifugal filtration tubes with 3K MWCO membranes. Thetubes were centrifuged at approximately 3000 RPM for 20 to 30 minutesand the concentrations of the solutions were checked using a NanoDrop2000 spectrophotometer. Solutions were concentrated to greater than 10mg/mL or 25 mg/mL and diluted to the appropriate concentrations usingthe appropriate buffer solution and the polysorbate concentration wasadjusted to 0.01% using a 1% solution of polysorbate 80. The solutionswere filtered through 0.22 μm syringe filters and filled into 3 mLglass, tubing vials at 0.25 mL and 0.8 mL per vial. The solutions werefreeze dried using a conservative cycle (Table 8.2) and placed onstability at 25° C. and 40° C. for up to 2 months.

TABLE 8.2 Conservative Lyophilization Cycle Used for Mannitol ContainingFormulations. Step Details Freezing Ramp 1° C./min to −40° C. IsothermalHold 120 min Annealing Ramp 1° C./min to −25° C., Hold 180 min PrimaryDrying −30° C., Hold until Pirani = CM Secondary Drying Ramp 0.5° C./minto 40° C., Hold until Pirani = CM

Samples of each solution prior to freeze drying were reserved forthermal analysis using DSC and FDM.

Additional thermal analyses and lyophilization cycle development studieswere completed using the buffer solutions prepared without the protein.The experiments were conducted to determine the minimum concentration ofarginine needed in the formulation to solubilize the protein while notinterfering with the crystallization of mannitol. Solubility studieswere conducted by the client to determine the minimum concentration ofarginine needed to solubilize the protein.₁ Experiments were conductedby Baxter to test the effect of arginine and mannitol concentration onthermal behavior, lyophilization cycle conditions, and cake appearance.The buffer solutions contained 10 mM of tris with 2% sucrose at pH 7.8.The arginine concentrations varied from 95 mM to 9.5 mM and the mannitolconcentrations were varied between 2% and 5%.

Good formulation candidates were identified based on thermal behavior,cake appearance, and short term accelerated stability data. The proposedformulation for further development contains 10-25 mg/mL r-Antidote, 10mM tris at pH 7.8, 45 mM arginine, 2% sucrose, 5% mannitol, and 0.01%polysorbate 80. Early studies used 0.2 mL to 1 mL per 3 mL vial. Theinitial stability study using a low arginine formulation was conductedusing a drug concentration of 25 mg/mL and lyophilized using aconservative cycle. The samples were placed on stability at 25° C. and40° C. for up to 3 months.

Cycles conducted to confirm the process used drug solution filled into10 mL vials at 5 mL per vial. The same vial and fill volume were usedfor studying the effect of moisture content during studies of secondarydrying. The formulations for these studies were prepared using drugsolution that was exchanged into the appropriate buffer using alaboratory-scale tangential flow filtration (TFF) unit. The TFF unit wasequipped with a holding vessel for the solution that was connected tothe tangential flow filter with tubing. The vessel was filled with drugsolution, exchanged into the appropriate buffer, and concentrated to 10to 25 mg/mL by filtering through a 10 KDa MWCO membrane. A sufficientquantity of 1% polysorbate 80 (PS80) was added to create a 0.01% PS80concentration. The final solution was filtered through a 0.22 μm syringefilter or vacuum filtration system.

The lyophilization cycles examined process parameters such as thecooling ramp rate and the ramp rate between primary and secondary dryingas well as the shelf temperature during annealing and primary drying. Aresidual moisture study was conducted by removing samples at thebeginning of secondary drying and after 4, 8, and 10 hours at 40° C. Asecond study was conducted by removing samples after 8 hours at 40° C.and after 1 and 2 hours at 50° C. The samples were tested for residualmoisture using Karl Fischer analysis and drying was considered completewhen the values for residual moisture reached a plateau. The effect ofthe residual moisture on the stability of the formulation was tested byremoving samples at times during secondary drying that corresponded tospecific residual moisture values. The samples were placed on stabilityat 40° C. for up to 2 months and at 50° C. for 1 week.

A lyophilization cycle design space was created for proposed drugproduct formulation containing 10 mg/mL r-Antidote, 10 mM tris, 45 mMarginine, 2% sucrose, 5% mannitol, and 0.01% polysorbate 80 at pH 7.8.The formulation was filled into 10 mL glass, tubing vials using 5 mLsolution per vial. Development of the design space requires knowledge ofthe equipment capability combined with the collapse temperature of theformulation and the heat transfer coefficient for the vial. The heattransfer coefficient for the vial was determined using the exact glass,tubing vial used for the product, filling the vials with water, andsubliming the ice using the shelf temperature intended for drying theproduct. Product temperature and mass flow data were collected whilevarying the chamber pressure from approximately 25 mTorr toapproximately 400 mTorr. Mass flow data were collected at each pressureusing tunable diode laser absorption spectroscopy (TDLAS) and the changein mass flow rate with pressure is used to calculate the heat transfercoefficient for the vial.

Results 1. Differential Scanning Calorimetry (DSC)

Individual solutions of each buffer component were prepared and testedusing DSC to determine the influence of each component on the thermalbehavior of the buffer formulation. Typically, the thermal behavior ofthe formulation is dictated by the component present at the highestconcentration. Changes in the thermal behavior can occur with theaddition of other excipients or the drug. For example, the addition ofsalts can decrease the Tg′ of amorphous materials in the formulation.The proposed drug formulation contains mannitol. Mannitol is added as anexcipient to lyophilized formulations to serve as a crystallizingbulking agent. Mannitol is amorphous when initially frozen in asolution. An annealing step is typically included during freezing toencourage crystallization of mannitol so that it can provide structurefor the cake. Other excipients and/or the active ingredient in aformulation can prevent or delay the crystallization of mannitol. Thestudies discussed in this section investigated the effects of tris,phosphate, and arginine on the crystallization of mannitol and thethermal behavior of the solution.

A 10 mM tris solution prepared at pH 7.8 was cooled at 1° C./min to −50°C. (FIG. 2) using DSC. The thermogram shows the crystallization exothermfor ice starting at approximately −20° C. followed by thecrystallization exotherm for tris at −32° C.

The crystallization exotherm is no longer present when 95 mM arginine isincluded in the formulation (FIG. 3). No thermal events besides themelting endotherm for ice were observed within the temperature range forthis study.

A Tg′ with a midpoint of approximately −42° C. is observed when the 10mM Tris, 95 mM arginine formulation contains 4% sucrose. The midpoint ofthe Tg′ for sucrose alone is typically around −33° C. The studydemonstrates that the tris/arginine mixture decreases the Tg′ forsucrose. A solution with a Tg′ below −40° C. is not a good candidate forlyophilization. It is difficult to maintain such a low producttemperature during primary drying. The addition of a crystallizingcomponent, such as mannitol, can provide structure and improve thechances of lyophilization as long as mannitol crystallizes before thestart of primary drying.

Mannitol was added to the formulation at 2% W/V and the sucroseconcentration was reduced to 2% so that the total sugar content in theformulation was maintained at 4%. A solution prepared with 10 mM tris,2% sucrose and 2% mannitol demonstrates that mannitol will begin tocrystallize at approximately −20° C. (FIG. 4). The crystallization ofmannitol is prevented when 95 mM arginine is added to the solution (FIG.5). Mannitol did not crystallize even when the frozen solution wasannealed at −20° C. for up to 5 hours (FIG. 6).

The same set of thermal analyses was conducted for solutions preparedwith 10 mM sodium phosphate to test the effect of the buffer on thethermal behavior of the formulation. Sodium phosphate crystallizedduring the cooling step (FIG. 7) when prepared as a 10 mM solution at pH7.8.

A mixture of 10 mM sodium phosphate with 95 mM arginine and 4% sucroseexhibits a Tg′ with a midpoint at approximately −38° C.

Similar to the tris solutions, phosphate solutions containing sucroseand mannitol exhibit a crystallization exotherm for mannitol (FIG. 8).The crystallization exotherm is not observed when 95 mM arginine isadded to the mixture (FIG. 9). Similar to the formulation prepared withtris, no crystallization exotherm was observed for mannitol even whenthe phosphate formulation was annealed at −20° C. for 5 hours.

The studies demonstrate that the addition of 95 mM arginine toformulations containing either tris or phosphate will drasticallydecrease the Tg′ for sucrose and will prevent the crystallization ofmannitol. The data demonstrated that a change to the formulation wasnecessary in order to encourage crystallization of mannitol for asuccessful lyophilized cake. At the time of this study, data suggestedthat either 95 mM arginine or 10 mM to 20 mM citrate were needed tomaintain the solubility of the protein. Therefore, studies wereconducted using solutions containing 10 mM or 20 mM citrate in 10 mMtris with 2% sucrose and 5% mannitol as an alternative to arginine inthe formulation. The mannitol concentration was increased and thesucrose concentration was decreased to increase the likelihood ofmannitol crystallization. Studies using 2% sucrose with 5% mannitolalong with arginine are described later in this report.

The solutions containing citrate were annealed at −25° C. Acrystallization exotherm was observed with an onset of 24 minutes in 10mM citrate at −25° C. (FIG. 10) and an onset of 30 minutes in 20 mMcitrate at −25° C. (FIG. 11).

2. Freeze Dry Microscopy (FDM)

The formulations prepared with 10 mM phosphate or 10 mM tris with 10mg/mL r-Antidote, 95 mM arginine, 2% sucrose, and 2% mannitol at pH 7.8were examined using FDM. Experiments conducted with the tris formulationshowed an onset of collapse for the formulation at approximately −34° C.when annealed at −25° C. for up to 3 hours.

The formulation containing 10 mM phosphate had a higher collapsetemperature. A consistent dry layer was observed at −32° C. and theonset of collapse was observed at −30° C.

The FDM data suggest that both formulations can be lyophilized usingconditions that are amenable to routine production. This does notcorrelate with data obtained using DSC. Experiments conducted using FDMutilize thin layers of solution between two glass coverslips in directcontact with a temperature controlled stage. These conditions suggest anease of drying and, therefore, failed to correlate with the DSC data,which were relied upon for subsequent testing given its relevance.

3. Lyophilization and Stability

The phosphate and tris formulations prepared with 10 mg/mL and 25 mg/mLr-Antidote, with 95 mM arginine, 2% sucrose, and 2% mannitol at pH 7.8were examined on stability as solutions and lyophilized samples. Eachsolution was filled into 3 mL vials at 0.20 mL per vial. A portion ofthe samples was stored at 5° C. and 25° C. for up to 2 weeks and theother portion of the samples was lyophilized using a conservative cycleand placed on stability at 25° C. for up to 3 months and at 40° C. forup to 2 months.

The samples were annealed at −25° C. for 1 hour before lyophilizing at−30° C. Secondary drying was conducted also using conservativeconditions with a 20° C. shelf temperature. A conservative,non-conventional cycle was used because little was known about thetemperature sensitivity of the protein. The lyophilization cycle wascompleted within approximately 21 hours. The vials were sealed withstoppers before removing from the lyophilizer, capped, and placed onstability.

The lyophilized cakes appeared acceptable with no evidence of collapseand reconstituted rapidly with purified water. A second study using thesame formulations without the drug was conducted concurrently to ensurethat the crystallization of mannitol, if it occurred, did not result inbreakage of vials. The placebo formulations were filled into 20 mL vialswith 10 mL of solution each. One full tray of vials was cooled to −40°C. at 1° C./min, held isothermally for 120 minutes, and then ramped to−25° C. at 1° C./min for 3 hours of annealing. A second set of vials wascooled to −25° C., held isothermally for 3 hours, cooled to −35° C. andthen transferred to the dryer containing the full tray of vials. Allvials were lyophilized at −30° C. and dried at 25° C. for secondarydrying. Collapse was observed in vials containing both formulations.

This suggests that the mannitol did not crystallize and supports theconclusion made during thermal analysis using DSC that arginine waspreventing the crystallization of mannitol. Therefore, the DSC andlyophilization data, given their relevance to the formulationdevelopment, rather than the FDM results, were relied upon for futureexperiments.

Studies described in the next example focused on the reduction ofarginine and its effect on the solubility of the protein andcrystallization of mannitol. The phosphate and tris formulationsprepared with 95 mM arginine described above remained on stability toprovide initial data.

No loss in concentration was observed in the solution samples whenstored at 5° C. and 25° C. for up to 2 weeks and there was no differencein concentration between the liquid and lyophilized samples at T0 (FIGS.12 and 13).

Similarly, no losses in concentration were observed in any of thelyophilized formulations stored at 25° C. for up to 3 months (FIG. 14)or at 40° C. for up to 2 months.

SEC data show that there were no losses in main peak when the solutionformulations were stored at 5° C. for up to 2 weeks. The percent mainpeak decreased by greater than 1% in 10 mg/mL samples and by greaterthan 3% in 25 mg/mL samples when stored at 25° C. for up to 2 weeks.

Therefore, although the chemical stability of the formulations appearsacceptable, changes to the formulation were necessary due to the poorphysical stability during lyophilization. Poor physical stability wasdemonstrated by the collapsed cakes observed for the placeboformulation. Data from DSC experiments suggest that decreasing thearginine concentration and increasing the mannitol concentration shouldencourage crystallization of mannitol and improve the physical stabilityof the lyophilized cake.

Example 8. Effects of Arginine and Mannitol Concentrations on theThermal Behavior and Appearance of the Lyophilized Samples

This example was conducted to investigate the effects of arginineconcentration and mannitol concentration on the thermal behavior andcake appearance using placebo formulations. The studies focused onplacebo formulations prepared with a tris buffer. Tris buffer was chosenbecause it is the buffer used to prepare the bulk drug solution andbecause there was no difference in the chemical stability of samplesprepared with tris and sodium phosphate.

The following studies examined using an arginine concentration range of9.5 mM to 95 mM and a mannitol concentration range of 2% to 5%.

1. Thermal Analysis

The goal of the thermal analysis experiments was to determine theconcentrations of arginine and mannitol that encouraged crystallizationof mannitol without substantially increasing the concentration of solidsin the formulation. High concentrations of solids can increase theresistance to mass transfer during lyophilization and create excessivelylong lyophilization cycles.

Arginine concentrations were reduced in the 10 mM tris, 2% sucrose, 2%mannitol, and 0.01% PS80 formulation while keeping the mannitolconcentration constant. Formulations were annealed at −15° C. to −25° C.for up to 5 hours to encourage crystallization. Crystallization ofmannitol was only observed when the arginine concentration was reducedto 9.5 mM and the annealing temperature was −22° C. or greater. Thecrystallization of mannitol began at the onset of annealing at −22° C.(FIG. 15).

The onset of crystallization for mannitol occurs after 30 minutes ofannealing at −25° C. when the concentration is increased to 4% and thearginine concentration is decreased from 95 mM to 47.5 mM (FIG. 16). Thelower annealing temperatures were investigated because changes to theappearance of the lyophilized cakes were observed when annealingoccurred at higher temperatures. Changes to the appearance included cakeshrinkage when annealing was conducted at −15° C.

The crystallization of mannitol, when using a concentration of 2% in theformulation, is delayed or prevented when the concentration of arginineis greater than 47.5 mM. The maximum concentration of arginine that canbe included in the formulation without affecting the crystallization ofmannitol is 47.5 mM. This statement was confirmed using lyophilizationexperiments that were conducted using the placebo formulation with 2%mannitol and 47.5 mM, 71 mM, or 85.5 mM arginine. Samples prepared with47.5 mM arginine were pharmaceutically acceptable, but samples preparedwith more arginine exhibited collapse. Increasing the concentration ofmannitol can increase the likelihood of crystallization. Annealing ofthe frozen solution is required to promote crystallization whenincreasing the concentration of mannitol to 4% and 5% when theconcentration of arginine was greater than 47.5 mM.

Mannitol readily crystallized in formulations containing 5% mannitol and47.5 mM arginine. A formulation containing 10 mM tris, 47.5 mM arginine,2% sucrose, 5% mannitol, and 0.01% PS80 was cooled slowly to −40° C. at1° C./min (FIG. 17). The crystallization exotherm for mannitol wasobserved during the cooling step when the formulation was cooled at 1°C./min.

A sample of the formulation was cooled quickly (cooled faster than 10°C./min) to −40° C. and then annealed at −25° C. (FIG. 18). Mannitolcrystallized after 23 minutes when the solution was annealed at −25° C.The experiments demonstrate that mannitol will readily crystallize inthe formulation as long as the arginine concentration is less than 47.5mM.

Thermal analysis data support that mannitol at a concentration of 4% orgreater will readily crystallize in a reasonable timeframe during alyophilization process if the arginine concentration is 47.5 mM or less.

2. Lyophilization

Lyophilization studies were conducted concurrently with thermal analysisexperiments. Placebo solutions prepared with 10 mM tris, 9.5 mM to 23.75mM arginine, 2% sucrose, and 2% to 4% mannitol, or 10 mM tris with 47.5mM arginine with or without 4% sucrose. A formulation containing 10 mMtris, 47.5 mM arginine, 2% sucrose, and 5% mannitol was also included.The solutions were filled into 20 mL vials using 3 mL solution per vial.A conservative, non-conventional lyophilization cycle was used todetermine if acceptable cakes could be produced. The samples were cooledto −20° C. at 1° C./min, annealed for 3 hours, cooled to −40° C. at 1°C./min, and held for 2 hours. The vacuum was initiated at 100 mTorr andthe shelf temperature was ramped to −30° C. at 0.5° C./min. Samples wereheld at −30° C. until the Pirani gauge value matched the capacitancemanometer (CM) value and then advanced to secondary drying at 25° C. at0.5° C./min. Secondary drying was complete when the Pirani gauge valuematched the CM value. Primary drying was complete after approximately 30hours and secondary drying required only a couple of hours.

Samples prepared with tris and arginine alone exhibited completecollapse and those that included 4% sucrose exhibited cake shrinkage.

All formulations containing 47.5 mM arginine or less and 2% to 5% ofmannitol appeared with acceptable cakes.

Studies were included to test the effect of annealing during the coolingramp using a 10 mL fill volume. The studies used samples prepared with10 mM tris, 2% sucrose, with 23.75 mM and 47.5 mM arginine, and 2% to 5%mannitol. The solution were cooled at 1° C./min to −25° C., held for 3hours, the vacuum was initiated at 100 mTorr, and the shelf temperaturewas increased to −20° C. at 0.5° C./min. The samples were dried at −20°C. and the shelf was warmed to 25° C. for secondary drying. All samplesappeared acceptable with no evidence of collapse.

The same formulations were used to examine the effect of cooling rate onthe appearance of the lyophilized cakes. One set of samples was cooledto −25° C. at 1° C./min and annealed for 3 hours. The second set ofsamples was cooled to −25° C. at 5° C./min and annealed for 3 hours. Thesets of samples were combined in a single dryer and lyophilized at −30°C. for primary drying followed by 25° C. for secondary drying.

All samples appeared acceptable with no evidence of collapse. The datasupport that cooling rates between 1° C./min and 5° C./min do not affectthe appearance of the samples.

Solubility studies conducted by the client supported that the proteinwould remain soluble in the solution if the concentration of argininewas 36 mM or greater in the pH range of 7.5 to 8.2. It was decided touse a solution that contained 45 mM arginine because it would ensurecomplete solubility of the protein while also being well below theconcentration that would prevent the crystallization of mannitol. Themannitol concentration was chosen as 5% to ensure that it readilycrystallized during the cycle. Therefore, the best formulation candidatewas 10 mM tris, 10 mg/mL or 25 mg/mL r-Antidote, 45 mM arginine, 2%sucrose, 5% mannitol, with 0.01% PS80 prepared at pH 7.8.

The lyophilization studies completed with placebo solutions demonstratedthat acceptable cakes could be produced when the arginine concentrationwas 47.5 mM or less with 2% mannitol or greater. The solutions werelyophilized using a shelf temperature as high as −20° C. with noevidence of collapse. Samples were annealed at −20° C. for 3 hoursduring the cooling step or after the freezing step at −40° C. with noeffect on the appearance of the cakes. The conservative and conventionalapproach is to first freeze samples at −40° C. followed by an annealingstep with primary drying. This approach was chosen for thelyophilization cycle. Primary drying was conducted after the annealingstep at −20° C. followed by increasing the shelf temperature at 0.5°C./min to 25° C. for secondary drying. Subsequent lyophilizationdevelopment studies focused on the appropriate secondary drying shelftemperature and duration.

The goals for development of the lyophilized formulation included (1)protein concentration of at least 10 mg/mL; (2) improved stability at2-8° C.; (3) reconstitution time of <5 min; and (4) robustlyophilization process.

Several rounds of formulation screening were performed to evaluate theeffect of individual variables on protein stability (both as alyophilized cake and in solution) and solubility at 5° C. A conservativelyophilization cycle was used during the formulation screening.lyophilization process development was performed in parallel.

The tests demonstrated that, in terms of protein concentration, higherconcentration (e.g., 25 mg/mL) solutions were less stable than lowerones (e.g., 10 mg/mL) after 2 days at room temperature (i.e., greaterincrease in total aggregates by SEC and in % beta peak by RP-HPLC). Theoptimum pH for r-Antidote stability (lyophilized product and insolution) was confirmed to be pH 7.80±0.3.

No significant difference in stability was observed between tris andphosphate buffer in presence of other stabilizing components (i.e.,sucrose and arginine).

In terms of stabilizer type and concentration, both 2% and 4% w/wsucrose provided a good stabilizing effect. An arginine concentrationof >36 mM is required to maintain solubility of the r-Antidote at >50mg/mL at 5° C., pH 7.80±0.3.

A crystalline component (bulking agent), mannitol, at a concentrationof >4% w/w (in presence of 10 mM tris, 2% w/w sucrose, 45 mM arginine)was important to avoid cake collapse during primary drying. Further, thepresence of a small amount of polysorbate 80 is critical to ensurer-Antidote stability in solution under shear conditions (shaking at roomtemperature)

The composition shown below exemplifies a suitable solution forlyophilization.

The lyophilized formulation improves the stability of the r-Antidotedrug product and can be stored at 2-8° C. The following table comparesthe compositions of the frozen liquid drug product and the reconstitutedlyophilized drug product. Examples of the composition of a 100 mg/vialand 400 mg/vial lyophilized drug product and example reconstitutedcompositions are presented.

Freeze drying microscopy was performed on two different formulations.Approximately 0.15 mL of solution was dispensed into a glass cell whichwas placed on a temperature-controlled freeze-drying stage.Thermocouples were placed onto the bottom and center of the cell tomonitor sample temperatures. The liquid was cooled at a rate of 0.5°C./min to −50° C., annealed at −20° C. for 1 hour, and refrozen to −50°C. The chamber was evacuated and heated at a rate of 0.5° C./min. Basedon this collapse temperature, combinations of freeze drying temperaturesand pressures that result in product temperatures below the collapsetemperature will produce a cake with no collapse. For example producttemperatures of up to 20° C. with 100 mTorr could be used to produce acake with no collapse.

Collapse Temperature Formulation (° C.) 10 mg/mL r-Antidote, 10 mM Tris,45 mM −15 L-Arginine HCl, 2% w/v Sucrose, 5% w/v Mannitol, 0.01%polysorbate 80, pH 7.8 20 mg/mL r-Antidote, 10 mM Tris, 45 mM −14L-Arginine HCl, 2% w/v Sucrose, 5% w/v Mannitol, 0.01% polysorbate 80,pH 7.8

After reconstitution r-Antidote for injection with SWFI, 50 mg/vial ispH 7.8, with an osmolality of ˜480 mOsm/kg. Therefore the reconstitutedDP is acceptable for intravenous administration.

r-Antidote BDS is formulated at 3.0 mg/mL in 10 mM Tris, pH 7.8±0.3, 4%sucrose, 95 mM arginine and stored frozen at −60° C. or colder. Themanufacture of r-Antidote for Injection consists of the thawing andpooling of the 3 mg/mL r-Antidote BDS, ultra filtration/diafiltrationagainst formulation buffer (10 mM tris, 2% sucrose, 5% mannitol, 45 mLarginine HCl, pH 7.8) to a final concentration of 10 mg/mL, spiking ofpolysorbate 80 to 0.01% w/w, aseptic filling, lyophilization,stoppering, capping and labeling into the r-Antidote for Injectioncontainer closure system.

The r-Antidote for injection manufacturing process utilizes procedureswhich were developed for the production of other sterile liquid drugproducts. The method of sterilization used to produce r-Antidote forInjection is 0.2 μm filtration. The r-Antidote is heat labile; therefore0.2 μm filtration is the most appropriate means of producing steriler-Antidote for injection.

The lyophilization process was developed using a rational approach basedon an understanding of the physical nature of the formulation componentsat different stages of the lyophilization cycle. Thermalcharacterization methods including differential scanning calorimetry(DSC) and freeze dry microscopy (FDM) were used to measure Tg′ (glasstransition temperature of the frozen concentrate) and Tc (collapsetemperature during primary drying). The cycle shown in the table belowwas selected for lyophilization of prototype batch J7128. The annealingstep allows crystallization of mannitol to ensure that producttemperature does not fall below collapse temperature during primarydrying. The primary drying temperature was selected to avoid cakecollapse with a reasonable duration of primary drying. The 2-stepsecondary drying condition was developed to produce a lyophilized DPwith a moisture level of <1% (see, e.g., Table 6).

1. An aqueous formulation, comprising from 10 mM to 55 mM arginine, from1% to 3% sucrose (w/v), from 2% to 8% mannitol (w/v) and at least 5mg/mL of a two-chain polypeptide comprising a first chain comprising theamino acid sequence of SEQ ID NO. 4, a second chain comprising the aminoacid sequence of SEQ ID NO. 5, and a disulfide bond between a firstCysteine residue at position 98 (Cys98) of SEQ ID NO. 4 and a secondCysteine residue at position 108 (Cys108) of SEQ ID NO. 5, wherein theformulation has a pH from 7.5 to
 8. 2. The formulation of claim 1,comprising from 40 mM to 50 mM arginine, from 1.5% to 2.5% sucrose(w/v), from 4.5% to 5.5% mannitol (w/v) and at least 10 mg/mL of thepolypeptide.
 3. The formulation of claim 2, comprising at least 18 mg/mLof the polypeptide.
 4. The formulation of claim 2, wherein thepolypeptide comprises an amino acid residue that is modified to bedifferent from natural amino acids.
 5. The formulation of claim 4,wherein residue Asp29 of the first chain is modified to(3R)-3-hydroxyAsp at Asp29.
 6. The formulation of claim 1, wherein thepolypeptide comprises at least an intra-chain disulfide bond for each ofthe first and second chains.
 7. The formulation of claim 1, comprisingat least about 10 mg/mL of the two-chain polypeptide.
 8. The formulationof claim 1, comprising at least about 20 mg/mL of the two-chainpolypeptide.
 9. An aqueous formulation, comprising from 10 mM to 55 mMarginine, from 1% to 3% sucrose (w/v), from 2% to 8% mannitol (w/v) andat least 5 mg/mL of a two-chain polypeptide comprising a first chaincomprising the amino acid sequence of SEQ ID NO. 4 or having at least90% sequence identity to SEQ ID NO. 4, a second chain of the amino acidsequence of SEQ ID NO. 5 or having at least 90% sequence identity to SEQID NO. 5, and a disulfide bond between a first Cysteine residue atposition 98 (Cys98) of SEQ ID NO. 4 and a second Cysteine residue atposition 108 (Cys108) of SEQ ID NO. 5, wherein the formulation has a pHfrom 7.5 to
 8. 10. The formulation of claim 9, wherein the two-chainpolypeptide has modifications to the Gla domain and the active site ascompared to the wild-type fXa protein, is able to bind to a fXainhibitor but does not assemble into a prothrombinase complex.
 11. Amethod of preparing a lyophilized formulation, comprising lyophilizingthe aqueous formulation of claim
 1. 12. A lyophilized compositionobtainable by lyophilizing the aqueous formulation of claim
 1. 13. Alyophilized composition comprising at least 10% (w/w) of a two-chainpolypeptide comprising a first chain of the amino acid sequence of SEQID NO. 4, a second chain of the amino acid sequence of SEQ ID NO. 5, anda disulfide bond between a first Cysteine residue at position 98 (Cys98)of SEQ ID NO. 4 and a second Cysteine residue at position 108 (Cys108)of SEQ ID NO. 5, and L-arginine HCl:sucrose:mannitol in a weight ratioof the range (0.5-1.4):(1-3):(2-8).
 14. The lyophilized composition ofclaim 13, comprising at least 18% (w/w) of the two-chain polypeptide.15. The lyophilized composition of claim 13, wherein the weight ratio ofL-arginine HCl:sucrose:mannitol is in the range of(0.9-1):(1.5-2.5):(4.5-5.5).
 16. The lyophilized composition of claim13, wherein the weight ratio of L-arginine HCl: sucrose:mannitol isabout 0.95:2:5.
 17. A method of reducing bleeding in a subjectundergoing anticoagulant therapy with a factor Xa inhibitor comprisingadministering to the subject a solution prepared by dissolving thelyophilized composition of claim 13 in an aqueous solvent.
 18. Themethod of claim 17, wherein the factor Xa inhibitor is apixaban,rivaroxaban or betrixaban.
 19. An aqueous formulation, comprising apolypeptide comprising the amino acid sequence of SEQ ID NO. 3 or anamino acid sequence having at least 95% sequence identity to SEQ ID NO.3, a solubilizing agent, a stabilizer, and a crystalline component,wherein the formulation does not collapse during lyophilization.
 20. Theformulation of claim 19, wherein the crystalline component is mannitolpresent in a concentration from 2% to 8% (w/v), the solubilizing agentis arginine and the stabilizer is sucrose.
 21. The formulation of claim19, further comprising a surfactant and a buffer.